Carrier polymer particle, process for producing the same, magnetic particle for specific trapping, and process for producing the same

ABSTRACT

Carrier polymer particles comprising organic polymer particles having a particle diameter of 0.1 to 20 micrometers and a saccharide with which the surface of the organic polymer particles is covered, the organic polymer particles and the saccharide being chemically bonded.

This application is a divisional application of U.S. Ser. No.13/025,273, which is a divisional application of U.S. application Ser.No. 11/914,986, filed on Mar. 3, 2008, which is a 371 ofPCT/JP06/309810, filed on May 17, 2006, and claims priority to JapanesePatent Application No. 2005-342167, filed on Nov. 28, 2005, JapanesePatent Application No. 2005-276052, filed on Sep. 22, 2005, and JapanesePatent Application No. 2005-147824, filed on Mar. 20, 2005.

TECHNICAL FIELD

The present invention relates to carrier polymer particles in which thesurface of organic polymer particles are covered with a saccharide, aprocess for producing the same, magnetic particles for specifictrapping, and a process for producing the same.

BACKGROUND ART

In recent years, attempts have been actively made in fields such as drugdiscovery to find molecules having specific interaction with a certainspecific molecule by utilizing intermolecular interaction. Specifically,immobilizing a molecule (probe molecule) having interaction on asupport, and trapping and purifying another molecule (target material)by utilizing a specific interaction is widely carried out.

For example, the discovery of the intracellular binding protein FKBP12of the immunosuppressant FK506 using an affinity resin (Nature, 341,758, 1989) has been known. A porous gel such as agarose is commonly usedas such affinity resin. However, when using a porous gel, the so-calledphenomenon nonspecific adsorption in which molecules other than thetarget molecule are adsorbed on the affinity resin arises and thus, theproblem that separation and purification of the target molecule isdifficult arises. A certain proportion among the probe molecules bondinternally to the porous gel and as a result of such probe moleculeshaving insufficient interaction with the target material, the problemarises that trapping efficiency of the target material is reduced.

As a solution to such nonspecific adsorption, microspheres made from astyrene/glycidyl methacrylate polymer, of which the surface is coveredwith glycidyl methacrylate, and a biologically-related material bondedto the polymer through a spacer have been proposed (JP-B-3086427 andJP-B-3292721). Also disclosed are particles having a hydrophilic spacerintroduced on the surface (WO 2004/025297 A1 and WO 2004/040305A1), andthe like. However, none of these have a sufficient effect in loweringnonspecific adsorption. Support particles having still smallernonspecific adsorption are desired. Also, the efficiency of trapping thetarget material of these particles is not sufficient.

On the other hand, as biologically-related material carrier polymerparticles which are sensitized by a chemical bonding method, carboxylgroup-modified polystyrene particles are widely used. However, since thepolystyrene particles generally have significant capability of adsorbingother biologically-related materials (nonspecific adsorption) which arenot target materials existing in the test sample, the performance of thesensitized particles is inhibited, posing a serious obstacle to use ofthe particles. In contrast, a blocking method, in which the surface ofthe particles is first sensitized with the target biologically-activematerial and a protein having little damage such as bovine serum albumin(BSA) is adsorbed on the remaining particle surface, has difficulty infully preventing nonspecific adsorption. Also, although it is known thatperformance of polystyrene particles as biologically-related materialcarrier particles can be improved by copolymerizing a styrene sulfonateor an acrylic ester having a polyalkylene oxide side chain representedby the formula (CH₂CH₂O)_(n) or (CH₂CHCH₃O)_(m) or by hydrolyzingfragments of persulfate initiator bonded to the particles by heattreatment in an alkaline aqueous solution after emulsion polymerizationof the particles, the nonspecific adsorption is not sufficientlyprevented. Also, the efficiency of trapping the target material of theseparticles is not sufficient.

DISCLOSURE OF THE INVENTION

An object of the invention is to provide carrier polymer particles whichhave very small nonspecific absorption of biological materials such asproteins and a process for producing the same.

Another object of the invention is to provide carrier polymer particleswhich have very small nonspecific absorption of biologically-relatedmaterials such as proteins and which have a high trapping efficiency ofthe target material, and a process for producing the same.

A further object of the invention is to provide magnetic particles forspecific trapping which have very small nonspecific absorption ofbiologically-related materials such as proteins, peptides, nucleicacids, and cells and a process for producing the same.

Carrier polymer particles according to a first aspect of the inventioncomprise organic polymer particles having a particle diameter of 0.1 to20 micrometers and a saccharide with which the surface of the organicpolymer particles is covered, wherein the organic polymer particles andthe saccharide are chemically bonded.

In the carrier polymer particles, the saccharide may be apolysaccharide.

In the carrier polymer particles, the saccharide may becarboxymethylated.

In the carrier polymer particles, the organic polymer particles and thesaccharide may be chemically bonded by a bonding group including atleast one of an amide bond and an ester bond.

A process for producing carrier polymer particles according to a secondaspect of the invention comprises covering the surface of organicpolymer particles having a particle diameter of 0.1 to 20 micrometerswith a saccharide by chemically bonding the organic polymers and thesaccharide.

When chemically bonding in the process for producing carrier polymerparticles, the organic polymer particles have a first functional groupand the saccharide has a second functional group, and the organicpolymer particles and the saccharide may be chemically bonded byreacting the first functional group and the second functional group.

In the process for producing carrier polymer particles, the firstfunctional group may be at least one functional group selected from thegroup consisting of a carboxyl group, an epoxy group, an amino group,and a tosyl group.

A process for producing carrier polymer particles according to a thirdaspect of the invention comprises:

-   -   covering organic polymer particles having a particle diameter of        0.1 to 20 micrometers and a functional group having reactivity        with a carboxyl group with a saccharide having a carboxyl group        by chemically bonding the organic polymer particles and the        saccharide; and    -   treating the organic polymer particles of which the surface has        been covered with the saccharide with a basic solution.

In the process for producing carrier polymer particles, the saccharidemay be a polysaccharide.

In the process for producing the carrier polymer particles, thechemically bonding may be achieved by a bonding group including at leastone of an amide bond and an ester bond.

In the process for producing carrier polymer particles, the functionalgroup having reactivity with the carboxyl group may be at least onefunctional group selected from the group consisting of an amino group, ahydroxyl group, and an epoxy group.

The process for producing the carrier polymer particles may furthercomprise chemically bonding a probe for specifically trapping a targetmaterial to the saccharide.

Magnetic particles for specific trapping according to a fourth aspect ofthe invention comprise magnetic particles having a particle diameter of0.1 to 20 micrometers and a saccharide, wherein the magnetic particlesand the saccharide are chemically bonded and a probe for specificallytrapping a target material is bonded to the saccharide.

The saccharide of the magnetic particles for specific trapping may be apolysaccharide.

The saccharide of the magnetic particles for specific trapping may becarboxymethylated.

The magnetic particles for specific trapping and the saccharide may bechemically bonded by a bonding group including at least one of an amidebond and an ester bond.

The magnetic particles of the magnetic particles for specific trappingare obtained by polymerization of a polymer layer on the magneticmaterial layer of mother particles comprising nuclear particles and amagnetic material layer formed on the surface of the nuclear particles,and the magnetic material layer may include at least one of Fe₂O₃ andFe₃O₄.

The probe of the magnetic particles for specific trapping may be atleast one selected from proteins, peptides, nucleic acids, glycosidecompounds, and synthetic chemical materials.

A process for producing magnetic particles for specific trappingaccording to a fifth aspect of the invention comprises:

-   -   chemically bonding magnetic particles having a particle diameter        of 0.1 to 20 micrometers and a saccharide; and    -   chemically bonding a probe for specifically trapping a target        material to the saccharide.

When chemically bonding the magnetic particles and the saccharide in theprocess for producing carrier polymer particles, the magnetic particleshave a first functional group and the saccharide has a second functionalgroup, and the magnetic particles and the saccharide may be chemicallybonded by reacting the first functional group and the second functionalgroup.

In the process for producing magnetic particles for specific trapping,the first functional group may be at least one functional group selectedfrom the group consisting of a carboxyl group, an epoxy group, an aminogroup, and a tosyl group.

The carrier polymer particles possess the characteristic of havinglittle nonspecific adsorption due to the organic polymer particleshaving a particle diameter of 0.1 to 20 micrometers, the saccharidecovering the surface of the organic polymer particles, and the organicpolymer and the saccharide are chemically bonded. Thus, the separationand the purification of target molecules can be easily carried out.

According to the process for producing carrier polymer particles,carrier polymer particles having little nonspecific adsorption and ahigh efficiency of trapping a target material can be obtained bycovering the organic polymer particles with a particle diameter of 0.1to 20 micrometers, which has a functional group having reactivity with acarboxyl group, with a saccharide having a carboxyl group by chemicallybonding the organic polymer particles and the saccharide, and treatingthe organic polymer particles, of which the surface has been coveredwith the saccharide, with a basic solution. Thus, the separation and thepurification of target molecules can be easily carried out.

Furthermore, the magnetic particles for specific trapping possess thecharacteristic of having little nonspecific adsorption, due to the useof the magnetic particles having a particle diameter of 0.1 to 20micrometers, inclusion of a saccharide, the magnetic particles arechemically bonded to the saccharide, and a probe for specificallytrapping a target material is chemically bonded to the saccharide. Thus,the separation and the purification of target molecules can be easilycarried out.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing an example of a process for producingcarrier polymer particles according to the first and second embodimentsof the invention.

FIG. 2 is a diagram showing an example of carrier polymer particlesaccording to the first and second embodiments of the invention.

FIG. 3 is a photograph showing the specific trapping evaluation resultsof the probe-bonded particles obtained in Experimental Examples 8 and 9,and Comparative Example 4 (electrophoresis pattern of the proteinsadsorbed on the probe bonding particles).

FIG. 4 is a photograph showing the specific trapping evaluation resultsof the probe-bonded particles obtained in Experimental Examples 10 and11, and Comparative Example 7 (electrophoresis pattern of the proteinsadsorbed on the probe bonding particles).

BEST MODE FOR CARRYING OUT THE INVENTION

The carrier polymer particles, the process for producing the same, themagnetic particles for specific trapping, and the process for producingthe same of the invention are explained in detail below.

1. First Embodiment 1-1. Carrier Polymer Particles

The carrier polymer particles according to the first embodiment of theinvention comprise organic polymer particles having a particle diameterof 0.1 to 20 micrometers and a saccharide with which the surface of theorganic polymer particles is covered. Also, the organic polymerparticles and the saccharide are chemically bonded in the carrierpolymer particles according to this embodiment. Although not limited, itis preferable that the chemically bonding is achieved by a bonding groupincluding at least one of an amide bond and an ester bond.

Although it is possible to use the carrier polymer particles accordingto the present embodiment as they are, they can also be used as adispersion liquid in which the particles are dispersed in a dispersionmedium in order to efficiently carry out a reaction with a compound. Asexamples of the dispersion medium, water; alcohols such as methanol,ethanol, propanol, isopropyl alcohol, n-butyl alcohol, isobutyl alcohol,sec-butyl alcohol, and tert-butyl alcohol; ethylene glycol derivativessuch as ethylene glycol, ethylene glycol monomethyl ether, ethyleneglycol monoethyl ether, ethylene glycol monopropyl ether, ethyleneglycol monobutyl ether, ethylene glycol monoethyl ether acetate,diethylene glycol monomethyl ether, diethylene glycol monoethyl ether,diethylene glycol dimethyl ether, and diethylene glycol diethyl ether;propylene glycol derivatives such as propylene glycol, propylene glycolmonomethyl ether, propylene glycol monoethyl ether, propylene glycolmonopropyl ether, propylene glycol monobutyl ether, and propylene glycolmonomethyl ether acetate; ketones such as acetone, methyl ethyl ketone,methyl isobutyl ketone, methyl amyl ketone, diisobutyl ketone, andcyclohexanone; esters such as ethyl acetate, butyl acetate, isobutylacetate, ethyl lactate, and gamma-butyl lactone; amides such asN,N-dimethylformamide, N,N-dimethylacetamide, and N-methylpyrrolidone;dimethyl sulfoxide; and aromatic hydrocarbons such as toluene and xylenecan be given.

The particle diameter of the carrier polymer particles according to thepresent embodiment are preferably 0.1 to 17 micrometers, and morepreferably 1 to 10 micrometers. When the particle diameter is less than0.1 micrometer, since separation using centrifugal separation or thelike takes a long time and separation of the washing solvent such aswater and the particles is insufficient, there are situations in whichthe removal of non-target molecules (for example, biologically-relatedmaterials such as proteins) is insufficient and thus, sufficientpurification is not possible. In contrast, since particles with adiameter exceeding 17 micrometers reduces the surface area of theparticles, there may be situations in which the trapped amount of thebiologically-related material such as proteins, which is the target, issmall.

Next, the constituting elements of the carrier polymer particlesaccording to the embodiment are explained in detail.

1-1-1. Organic Polymer Particles

The average particle diameter of the organic polymer particles used inthe present embodiment is 0.1 to 20 micrometers, more preferably 0.3 to15 micrometers, and most preferably 1 to 10 micrometers. Also, thecoefficient of variation of the organic polymer particles used in theinvention is normally 30% or less, preferably 20% or less, and morepreferably 10% or less.

In the embodiment, the organic polymer particles may be used as baseparticles of the carrier polymer particles according to the embodiment.Organic polymer particles are suitable as base particles, since it iseasy to cover the surface of organic polymer particles with a saccharidewhich is bonded by chemically bonding. Also, magnetic particles may beused as the organic polymer particles.

As explained above, when the carrier polymer particles according to theembodiment are dispersed in a solvent, nonspecific adsorption ofbiologically-related materials such as proteins increases if the organicpolymer particles are dispersed in a dispersion medium or the organicpolymer particles swell by the solvent. For this reason, it is desirablethat the organic polymer particles do not dissolve in the dispersionmedium. Here, an aqueous medium may be used as the dispersion medium,for example. Here, aqueous medium means water or a mixture of water anda solvent which are dissolved in water (for example, alcohols andalkylene glycol derivatives).

Vinyl polymers are particularly preferable as the polymer constitutingthe organic polymer particles. As examples of the vinyl monomersconstituting the vinyl polymer, aromatic vinyl monomers such as styrene,alpha-methyl styrene, halogenated styrene, and divinylbenzene; vinylesters such as vinyl acetate and vinyl propionate; unsaturated nitrilessuch as acrylonitrile; ethylenic unsaturated carboxylic acid alkylesters such as methyl acrylate, ethyl acrylate, ethyl methacrylate,butyl acrylate, butyl methacrylate, 2-ethylhexyl acrylate, 2-ethylhexylmethacrylate, lauryl acrylate, lauryl methacrylate, cyclohexyl acrylate,and cyclohexyl methacrylate; polyfunctional (meth)acrylates such asethylene glycol diacrylate, ethylene glycol dimethacrylate, trimethylolpropane triacrylate, and trimethylol propane trimethacrylate; and(meth)acrylates having a functional group such as glycidyl acrylate,glycidyl methacrylate, 2-hydroxyethyl acrylate, and 2-hydroxyethylmethacrylate can be given. The vinyl polymer may be a homopolymer or maybe a copolymer comprising two or more monomers selected from theabove-mentioned vinyl monomers. Also, a copolymer of the above-mentionedvinyl monomers and copolymerizable monomers such as conjugated diolefinssuch as butadiene and isoprene, acrylic acid, methacrylic acid, itaconicacid, acrylamide, methacrylamide, N-methylol acrylamide, N-methylolmethacrylamide, diallyl phthalate, allyl acrylate, and allylmethacrylate can also be used.

The magnetic particles are general particulate materials which can bemagnetically collected and include fine particles of a magneticmaterial. When the organic polymer particles used in this embodiment aremagnetic particles, the carrier polymer particles according to theembodiment can be used as magnetic particles usable in applicationsdescribed later as examples.

If the particle diameter of the magnetic particles is less than 0.1micrometer, it may take a long time for separation and purificationusing magnetism; and if more than 20 micrometers, the amount of trappedtarget material such as proteins may be small due to the surface areabecoming smaller.

Although the internal composition of the magnetic particles may behomogeneous, most magnetic materials making up the homogeneous magneticparticles with a particle diameter in the above-mentioned preferablerange are paramagnetic. If repeatedly separated and purified bymagnetism, the magnetic particles may lose their capability of beingredispersed in dispersion media. For this reason, it is preferable thatthe magnetic particles have a heterogeneous internal compositioncontaining fine particles of a magnetic material exhibiting smallresidual magnetization. As the inner structure of the magnetic particleshaving such a heterogeneous internal composition, a structure in whichthe fine particles of a magnetic material are dispersed in a continuousphase of a non-magnetic material such as a polymer, a structureconsisting of a secondary aggregate of fine particles of a magneticmaterial as a core and a non-magnetic material such as a polymer layeras a shell, a structure consisting of a non-magnetic material such as apolymer (non-magnetic nuclear particles) as a core and a secondaryaggregate material of fine particles of a magnetic material as a shell,and the like can be given. As the polymer which is included in themagnetic particles, the polymers described above as the polymer formingthe organic polymer particles may be used. In the case of the structurein which the inner structure consists of a core of a non-magneticmaterial such as a polymer (non-magnetic nuclear particles) and a shellof a secondary aggregate of fine particles of a magnetic material, it ispreferable that a polymer layer be further formed on the outermostlayer. As the polymer which is used for the outermost layer, thepolymers described above as the polymer forming the organic polymerparticles as the base particles may be used.

The organic polymer particles of this embodiment may be produced by ageneral method such as emulsion polymerization, soap-freepolymerization, and suspension polymerization. When the organic polymerparticles are magnetic particles, such organic polymer particles may beproduced by, for example, mixing the non-magnetic material nuclearparticles with the fine particles of a magnetic material and causing thefine particles of a magnetic material to be physically adsorbed on thesurface of the non-magnetic material nuclear particles. In thisembodiment, “physical adsorption” refers to adsorption not involving achemical reaction. As the principle of “physical adsorption”,hydrophobic/hydrophobic adsorption, molten bonding or adsorption, fusionbonding or adsorption, hydrogen bonding, Van der Waals bonding, and thelike can be given, for example.

More specifically, the organic polymer particles may be obtained by, forexample, suspension polymerization of the above vinyl monomer or polymerbulk shattering. For example, the organic polymer particles can beobtained by a two-stage swelling polymerization method using seedparticles described in JP-B-57-24369, the polymerization methoddescribed in J. Polym. Sci., Polymer Letter Ed., 21, 937 (1963), and themethods described in JP-A-61-215602, JP-A-61-215603, and JP-A-61-215604.

The magnetic particles can also be prepared by a method utilizinghydrophobic/hydrophobic adsorption as mentioned above. For example, amethod selecting non-magnetic nuclear particles and fine particles of amagnetic material, each having a hydrophobic or hydrophobized surface,and dry-blending these non-magnetic nuclear particles and fine particlesof a magnetic material, and a method sufficiently dispersing thenon-magnetic nuclear particles and the fine particles of a magneticmaterial in a solvent (such as toluene or hexane) with gooddispersibility without damaging both particles, followed by vaporizationof the solvent while mixing can be given. Alternatively, the magneticparticles may be produced by a method realizing complexing onnon-magnetic nuclear particles and fine particles of a magnetic materialby physically applying a strong external force. As examples of themethod for physically applying a strong force, a method using a mortar,an automatic mortar, or a ball mill; a blade-pressuring type powdercompressing method; a method utilizing a mechanochemical effect such asa mechanofusion method; and a method using an impact in a high-speed airstream such as a jet mill, a hybridizer, or the like can be given. Inorder to efficiently produce a firmly bound complex, a strong physicaladsorption force is desirable. As the method, stirring using a vesselequipped with a stirrer at a peripheral velocity of stirring blades ofpreferably 15 msec or more, more preferably 30 msec or more, and stillmore preferably from 40 to 150 msec can be given. If the stirring bladeperipheral velocity is less than 15 msec, sufficient energy for causingfine particles of a magnetic material to be adsorbed on the surface ofnon-magnetic nuclear particles may not be obtained. Although there areno specific limitations to the upper limit of the peripheral speed ofthe stirring blades, the upper limit of the peripheral speed isdetermined according to the apparatus to be used, energy efficiency, andthe like.

1-1-2. Saccharide

As examples of the saccharide used for the carrier polymer particlesaccording to this embodiment, monosaccharides, such as furanoses such asfructose, arabinose, xylose, ribose, and deoxyribose, pyranoses such asglucose, mannose, and galactose, and septanoses; disaccharides such astrehalose, lactose, kojibiose, nigerose, maltose, isomaltose, sophorose,laminaribiose, cellobiose, and gentiobiose; and polysaccharides such asstarch, amylose, amylopectin, dextrin, glycogen, cyclodextrin,cellulose, agarose, alginic acid, inulin, glucomannan, chitin, chitosan,and hyaluronic acid can be given. In order to cover the surface oforganic polymer particles by the chemical bond of the organic polymerparticles and the saccharide, a polysaccharide with a high molecularweight is preferable from the viewpoint of coating efficiency. Asaccharide of which at least a part of the functional group (such as ahydroxyl group, an amino group, and a carboxyl group) has been modified,such as carboxymethylcellulose and carboxymethyldextran, may be used.The modification may be made in multiple stages, if necessary. Morepreferably, a carboxymethylated saccharide such ascarboxymethylcellulose or carboxymethyldextran can be used.

1-2. Process for Producing Carrier Polymer Particles

The process for producing carrier polymer particles of this embodimentcomprises covering the surface of organic polymer particles having aparticle diameter of 0.1 to 20 micrometers with a saccharide bychemically bonding the organic polymers and the saccharide.

In this embodiment, a general chemical reaction may be used forchemically bonding the organic polymer particles with a saccharidewithout any particular limitations.

FIG. 1 is a diagram showing the process for producing the carrierpolymer particles according to this embodiment. FIG. 2 is a diagramshowing the process for producing the carrier polymer particlesaccording to this embodiment, wherein a carrier polymer particle 10 ofthe embodiment produced by the process shown in FIG. 1 is shown.

For example, as shown in FIG. 1, the organic polymer particle 11 usedfor producing the carrier polymer particles according to this embodimentmay have a plurality of functional groups 13 (first functional groups)on the surface. The first functional groups 13 may be functional groupsintroduced when the particle shape of the organic polymer particle 11 isformed or functional groups obtained by converting the functional groupsafter the particle shape of the organic polymer particle 11 has beenformed. The conversion of functional groups may be carried out two ormore times. Although not particularly limited, when the functional groupintroduced when forming the particle shape of the organic polymerparticle 11 is an epoxy group, an amino group produced by reacting theepoxy group with a large excess amount of ammonia or an appropriatediamine compound may be the first functional group, or when thefunctional group introduced when forming the particle shape of theorganic polymer particle 11 is a hydroxyl group, for example, an aminogroup produced by converting the hydroxyl group into a tosyl group andreacting the tosyl group with a large excess amount of an appropriatediamine compound may be the first functional group 13. For example, inthe organic polymer particles Am-1 to Am-5 respectively obtained in thelater-described Experimental Examples 1 to 3, and Experimental Examples5 to 6, the first functional group 13 may be the amino group.

The saccharide 12 used for producing the carrier polymer particlesaccording to this embodiment may have a plurality of functional groups(second functional groups) 14 in the molecule. The functional group maybe produced by converting the functional group of a saccharide.

As the functional group which can be used as the first functional group13 and/or the second functional group 14, a carboxyl group, a hydroxylgroup, an epoxy group, an amino group, a mercapto group, a vinyl group,an allyl group, an acrylic group, a methacryl group, a tosyl group, anazido group, and the like can be given. The first functional group 13and the second functional group 14 are reactive with each other.Although not particularly limited, when the first functional group 13 isan epoxy group, for example, the second functional group 14 may be anamino group, or when the first functional group 13 is an amino group,for example, the second functional group 14 may be a carboxyl group.

It is possible to chemically bond the organic polymer particle 11 with asaccharide 12 by reacting the first functional group 13 and the secondfunctional group 14 (see FIG. 2), whereby the carrier polymer particle10 according to this embodiment can be obtained.

After preparation according to the above-described process, the carrierpolymer particles of this embodiment can be used as carrier particlesafter adjusting the pH and washing the surface by a purification processsuch as dialysis, ultrafiltration, and centrifugation, as required.

1-3. Application

The carrier polymer particles of this embodiment are used as chemicalbonding carrier polymer particles in the drug discovery field and alsoas chemical bonding carrier polymer particles for diagnostic agent.

More particularly, the carrier polymer particles of this embodiment canbe used for selecting and purifying proteins and the like exhibitingspecific interactions with a chemical compound to be analyzed byimmobilizing such a chemical compound to be analyzed by chemical bondingand analyzing and/or measuring the specific interactions usingintermolecular interactions with the proteins and the like.

In addition, the carrier polymer particles of this embodiment can alsobe used as biologically-related material carrier polymer particles forsensing proteins such as an antibody, an antigen, an enzyme, and ahormone, nucleic acids such as DNA and RNA, and biologically-relatedglycoside compounds (hereinafter referred to collectively as“biologically-related material”) on the surface of particles by achemical bonding method.

The applications of the carrier polymer particles of this embodiment arenot limited to the above-mentioned applications of chemical bondingcarrier polymer particles in the drug discovery field and chemicalbonding carrier polymer particles for diagnostic drugs. The carrierpolymer particles can be used in a wide variety of fields such asbiologically-related fields, paints, papers, electrophotography,cosmetics, medical supplies, agricultural chemicals, foods, andcatalysts.

2. Second Embodiment 2-1. Process for Producing Carrier PolymerParticles

The process for producing carrier polymer particles according to thesecond embodiment comprises covering organic polymer particles with aparticle diameter of 0.1 to 20 micrometers, which has a functional grouphaving reactivity with a carboxyl group, with a saccharide having acarboxyl group by chemically bonding the organic polymer particles andthe saccharide (a first step), and treating the organic polymerparticles, of which the surface has been covered with the saccharide,with a basic solution (a second step).

The first step and second step in the process for producing the carrierpolymer particles according to the embodiment will be explained indetail.

2-1-1. First Step

The functional group which has reactivity with a carboxyl group whichexists in the organic polymer particles in the first step of thisembodiment is at least one functional group selected from the groupconsisting of an amino group, a hydroxyl group, and an epoxy group,preferably at least one functional group selected from the groupconsisting of an amino group and a hydroxyl group, and more preferablyan amino group.

In the first step of this embodiment, a general chemical reaction may beused for chemically bonding the organic polymer particles to asaccharide without any particular limitations. The particles may bechemically bonded to the saccharide by a bonding group including atleast one of an amide bond and an ester bond.

FIG. 1 is a diagram showing the first step of the process for producingcarrier polymer particles according to this embodiment, and FIG. 2 is adiagram showing a example of the carrier polymer particles produced inthe first step.

For example, as shown in FIG. 1, the organic polymer particle 11 usedfor producing the carrier polymer particles according to this embodimenthas functional groups 13, which are reactive with a carboxyl group, onthe surface. The functional groups 13 which are reactive with a carboxylgroup may be introduced when the particle shape of the organic polymerparticle 11 is formed, or may be obtained by converting a certain groupafter the particle shape of the organic polymer particle 11 has beenformed. The conversion of functional groups may be carried out two ormore times. For example, when the functional group introduced whenforming the shape of the organic polymer particle 11 is an epoxy group,an amino group produced by reacting the epoxy group with a large excessamount of ammonia or an appropriate diamine compound may be thefunctional groups 13 which are reactive with a carboxyl group, or whenthe functional group introduced when forming the shape of the organicpolymer particle 11 is a hydroxyl group, an amino group produced byconverting the hydroxyl group into a tosyl group and reacting the tosylgroup with a large excess amount of an appropriate diamine compound maybe the functional groups 13 which are reactive with a carboxyl group.For example, in the organic polymer particles Am-1 to Am-5 respectivelyobtained in the later-described Experimental Examples 1 to 3, andExperimental Examples 5 to 6, the functional group 13 which is reactivewith a carboxyl group is the amino group.

The saccharide 12 used for producing the carrier polymer particlesaccording to this embodiment may have one carboxyl group 14 or aplurality of carboxyl groups 14 in the molecule. The carboxyl group 14may be a group produced by converting a specific functional group of thesaccharide 12.

As an example of the group which can be used as the functional group 13reactive with a carboxyl group, an amino group can be given. Asmentioned above, the amino group may be converted from another group(such as a hydroxyl group or an epoxy group) which is introduced whenthe particle shape of the organic polymer particles are formed. Thefunctional group 13 reactive with a carboxyl group and the carboxylgroup 14 are reactive with each other. Although not limited, when thefunctional group 13 having reactivity with a carboxyl group is at leastone selected from the group consisting of an amino group, a hydroxylgroup, and an epoxy group, for example, this functional group 13 hasreactivity with the carboxyl group 14.

In FIG. 1, the organic polymer particle 11 and the saccharide 12 canchemically bond by reacting the functional group 13 having reactivitywith a carboxyl group and the carboxyl group 14.

A general method can be used for this chemical bonding without specificlimitations. For example, when the group having reactivity with acarboxyl group is an epoxy group, an ester can be produced by directlyreacting them. When the group having reactivity with a carboxyl group isa hydroxyl group, an esterification method using various condensingagents can be used (The 4th edition of Experimental Chemistry LectureVol. 22, pp 45-47, 1992). When the group having reactivity with acarboxyl group is an amino group, an amidation method using variouscondensing agents commonly used in organic synthesis (The 4th edition ofExperimental Chemistry Lecture Vol. 22, pp 139-144, 1992) and variousmethods used for forming a peptide bond in peptide synthesis (The 4thedition of Experimental Chemistry Lecture Vol. 22, pp 259-271, 1992) canbe used.

After the organic polymer particle 11 and the saccharide 12 have beenchemically bonded and the surface of the organic polymer particle 11 iscovered with the saccharide 12 (see FIG. 2), an excess amount of thesaccharide existing in the reaction system (not shown in the drawing)may be physically adsorbed in the saccharide 12 which chemically bondedto the organic polymer particle 11 by hydrogen bonds and the likebetween carboxyl group and carboxyl group, carboxyl group and hydroxylgroup, and/or hydroxyl group and hydroxyl group. In order tosufficiently cover the surface of the organic polymer particle 11 with achemically bonded saccharide 12, it is necessary to use an excess amountof a saccharide. Therefore, occurrence of physical adsorption ofsaccharide 12 to a certain degree is inevitable. If the target materialis separated and purified by using the organic polymer particle 11 inwhich a saccharide is physically adsorbed, problems such as reducedutilization efficiency of the carboxyl group 14, an increase innonspecific adsorption due to the surface of the particle 10 partiallymade porous, and detachment of the once-trapped target material togetherwith the physically adsorbed saccharide during separation andpurification operations may occur.

2-1-2. Second Step

In the second step of this embodiment, the saccharide physicallyadsorbed on the surface of the organic polymer particle 11 in the firststep can be extracted by a basic solution by treating the organicpolymer particle 11 of which the surface was covered by saccharide 12with the basic solution. In the second step, by treating the organicpolymer particle 11 with a sufficient amount of basic solution, onlychemically bonded saccharide 12 finally remains on the surface of theorganic polymer particle 11 (see FIG. 2), whereby the above-mentionedproblems caused by the physically adsorbed saccharide can be overcome.The carrier polymer particles comprising organic polymer particle 11 andthe saccharide 12 covering the surface of the organic polymer particle11, from which physically adsorbed saccharide has been removed, can beobtained by the above steps.

The basic solution used here is not particularly limited insofar as thesolution can extract the physically adsorbed saccharide. For example,alkaline aqueous solutions such as a sodium hydroxide aqueous solution,a potassium hydroxide aqueous solution, a lithium hydroxide aqueoussolution, a sodium carbonate aqueous solution, a sodium hydrogencarbonate aqueous solution, a potassium carbonate aqueous solution, apotassium hydrogen carbonate aqueous solution, a lithium carbonateaqueous solution, ammonia water, and a hydroxyl tetramethylammoniumaqueous solution; and aqueous solutions of water-soluble organic aminescan be given.

The concentration of the basic aqueous solution used here is usually0.001 M or more. The treating temperature is usually 0 to 50° C., andpreferably 0 to 30° C.

Although inferior to the basic solution in respect of efficiency, thephysically-adsorbed saccharide can also be extracted by treating with anappropriate electrolytic solution.

2-1-3. Third Step

The process for producing the carrier polymer particles may furthercomprise a step of chemically bonding the saccharide to a probe forspecifically trapping a target material (a third step). The particlesobtained by the third step have a probe to specifically trap a targetmaterial chemically bonded to a saccharide (such particles are hereinreferred to as “probe-bonded particles). Although not specificallylimited, the saccharide and the probe may bond via a chemical bond suchas an —O— bond, an —S— bond, an —SO— bond, an —SO₂— bond, a —CO— bond, a—CO₂— bond, an —NR¹— bond (wherein R^(I) is an alkyl group or H), an—N⁺R²R³— bond (wherein R² and R³ are individually an alkyl group or H),an —NHCO— bond, or a —PO₂— bond. The saccharide and the probe can bechemically bonded by, for example, chemically reacting a functionalgroup in the saccharide with a functional group in the probe.

The functional group in the saccharide and the functional group in theprobe are not specifically limited. As examples, groups such as ahydroxyl group, an acyl group, a mercapto group, an amino group, anaminoacyl group, a carbonyl group, a formyl group, a carboxyl group, anamide group, a sulfonic group, a phosphate group, an epoxy group, atosyl group, an azido group, a vinyl group, and an allyl group can begiven.

In this embodiment, the term “target material” refers to a target to betrapped by the probe-bonded particles according to this embodiment. Asan example of the target material, a biologically-related material canbe given. In the embodiment, the term “biologically-related material”refers to all materials relating to biological bodies. As examples ofthe biologically-related material, materials contained in biologicalbodies, materials derived from materials contained in biological bodies,and materials which can be used in biological bodies can be given.

More specific examples of the biologically-related materials include,but are not limited to, proteins (such as an enzyme, an antibody, and anacceptor), peptides (such as glutathione and RGD peptides), nucleicacids (such as DNA and RNA), carbohydrates, lipids, and other cells andmaterials (such as various blood-originating materials and variousfloating cells containing various blood cells such as platelets,erythrocytes, and leukocytes).

When the probe is a protein, for example, the probe can be chemicallybonded to the saccharide by, for example, reacting a functional group inthe protein (for example, an amino group or a carboxyl group) with afunctional group in the saccharide (for example, a carboxyl group, ahydroxyl group, or an amino group). In this instance, the probe and thesaccharide can be bonded through an amide bond or an ester bond. Whenthe probe is a nucleic acid, for example, the probe can be chemicallybonded to the saccharide by, for example, reacting a functional group inthe nucleic acid (for example, a phosphoric acid group) with afunctional group in the saccharide (for example, a hydroxyl group). Inthis instance, the probe and the saccharide can be bonded through aphosphodiester bond.

Although not particularly limited, the probe which can be used with theparticles for specific trapping includes, for example, a protein (forexample, an antibody, an antigen, an enzyme, an acceptor, and ahormone), a peptide, a nucleic acid (for example, DNA and RNA), aglycoside compound, and a synthetic chemical material (for example, apharmaceutical candidate compound).

When the probe is an antibody (or an antigen), the target material maybe an antigen (or an antibody) which specifically bonds to the antibody(or the antigen).

When the probe is a nucleic acid, the target material may be a nucleicacid which specifically bonds to the nucleic acid. When the probe is anenzyme, an acceptor, or a hormone, the target material may be a chemicalcompound which specifically bonds to the enzyme, the acceptor, or thehormone.

2-1-4. Materials Used for Producing Carrier Polymer Particles of thisEmbodiment

Next, the materials used for producing the carrier polymer particlesaccording to this embodiment are explained in detail.

2-1-4A. Organic Polymer Particles

The average particle diameter of the organic polymer particles used forproducing the carrier polymer particles of this embodiment is 0.1 to 20micrometers, more preferably 0.3 to 15 micrometers, and most preferably1 to 10 micrometers. Also, the coefficient of variation of the organicpolymer particles used in the embodiment is normally 30% or less,preferably 20% or less, and more preferably 10% or less.

In this embodiment, the organic polymer particles may be used as baseparticles of the carrier polymer particles produced according to thisembodiment. Organic polymer particles are suitable as base particles,since it is easy to cover the surface of organic polymer particles witha saccharide which is bonded by chemical bonding. Also, magneticparticles may be used as the organic polymer particles.

As explained above, when the carrier polymer particles producedaccording to this embodiment are dispersed in a solvent, nonspecificadsorption of biologically-related materials such as proteins increasesif the organic polymer particles are dispersed in a dispersion medium orthe organic polymer particles swell by the solvent. For this reason, itis desirable that the organic polymer particles do not dissolve in thedispersion medium. An aqueous medium may be used as the dispersionmedium, for example. The aqueous medium means water or a mixture ofwater and a solvent which dissolves in water (for example, alcohols andalkylene glycol derivatives).

As the organic polymer particles, polymers used for the organic polymerparticles of the first embodiment can be used. In addition, the processdescribed in the first embodiment can be used. Vinyl polymers areparticularly preferable as the polymer constituting the organic polymerparticles.

As the magnetic particles, those given as the magnetic particles infirst embodiment may be used.

2-1-4B. Saccharide

As examples of the saccharide having a carboxyl group used for producingthe carrier polymer particles according to this embodiment, saccharidesobtained by chemically modifying at least a part of the functionalgroups (for example, a hydroxyl group and an amino group) in themolecule of a saccharide by introducing a carboxyl group, for example,carboxymethylcellulose, carboxymethyldextran, and polysaccharidesoriginally possessing a carboxyl group (such as alginic acid andhyaluronic acid) can be given. Such a saccharide to be modified byintroducing a carboxyl group includes furanoses such as fructose,arabinose, xylose, ribose, and deoxyribose; pyranoses such as glucose,mannose, and galactose; monosaccharides such as septanoses;disaccharides such as trehalose, lactose, kojibiose, nigerose, maltose,isomaltose, sophorose, laminaribiose, cellobiose, and gentiobiose; andpolysaccharides such as starch, amylose, amylopectin, dextrin, glycogen,cyclodextrin, cellulose, agarose, inulin, glucomannan, chitin, andchitosan.

A general chemical reaction may be used for chemically modifying thesaccharide to introduce a carboxylic group without any particularlimitations. The chemical modification may be carried out in two or morestages as required. In order to cover the surface of organic polymerparticles by the chemical bond of the organic polymer particles and thesaccharides, a polysaccharide with a high molecular weight is preferablefrom the viewpoint of coating efficiency. Carboxymethylcellulose andcarboxymethyldextran are particularly preferable as the saccharidehaving a carboxyl group.

2-1-5. Carrier Polymer Particles

The particle diameter of the carrier polymer particles produced by thisembodiment is preferably 0.1 to 17 micrometers, and more preferably 1 to10 micrometers. When the particle diameter is less than 0.1 micrometer,since separation using centrifugal separation or the like takes a longtime and separation of the washing solvent such as water and theparticles is insufficient, there are situations in which the removal ofnon-target molecules (for example, biologically-related materials suchas proteins) is insufficient and thus, sufficient purification is notpossible. In contrast, since particles with a diameter exceeding 17micrometers reduces the surface area of the particles, there may besituations in which the trapped amount of the biologically-relatedmaterial such as a protein, which is the target, is small.

After preparation according to the above-described process, the carrierpolymer particles of this embodiment can be used as carrier particlesafter adjusting the pH and washing the surface by purificationprocessing such as dialysis, ultrafiltration, and centrifugation, asrequired.

Although it is possible to use the carrier polymer particles produced bythis embodiment as they are, they can also be used as a dispersionliquid in which the particles are dispersed in a dispersion medium inorder to efficiently carry out a reaction with a compound. As adispersion medium, those given as the dispersion medium in firstembodiment may be used.

In addition, the carrier polymer particles of the embodiment may beprobe-bonded particles in which a probe to specifically trap a targetmaterial chemically bonds to the saccharide. The probe may be chemicallybonded to the saccharide by the above-described third step. The particlediameter of the probe-bonded particles is preferably from 0.1 to 20micrometers, more preferably from 0.3 to 17 micrometers, and still morepreferably from 0.5 to 10 micrometers.

2-2. Application

The carrier polymer particles produced by this embodiment may be used aschemical bonding carrier polymer particles in the drug discovery fieldand also as chemical bonding carrier polymer particles for diagnosticdrugs.

More particularly, the carrier polymer particles produced by thisembodiment can be used for selecting and purifying a target material(such as proteins) exhibiting specific interactions with a chemicalcompound to be analyzed by immobilizing the chemical compound to beanalyzed by chemical bonding.

In addition, the carrier polymer particles produced by this embodimentcan also be used as biologically-related material carrier polymerparticles for sensing proteins such as an antibody, an antigen, anenzyme, and a hormone; nucleic acids such as DNA and RNA; andbiologically-related glycoside compounds (hereinafter referred tocollectively as “biologically-related material”) on the surface ofparticles by a chemical bonding method.

The applications of the carrier polymer particles produced by thisembodiment is not limited to the above-mentioned applications ofchemical bonding carrier polymer particles in the drug discovery fieldand chemical bonding carrier polymer particles for diagnostic drugs. Thecarrier polymer particles can be used in a wide variety of fields suchas biologically-related fields, paints, papers, electrophotography,cosmetics, medical supplies, agricultural chemicals, foods, andcatalysts.

3. Third Embodiment 3-1. Magnetic Particles for Specific Trapping

The magnetic particles for specific trapping according to the thirdembodiment of the invention comprise magnetic particles and asaccharide. The saccharide may cover the surface of the magneticparticles.

The magnetic particles chemically bond with the saccharide in themagnetic particles for specific trapping. Although not specificallylimited, the chemical bond of the magnetic particles and the saccharideis preferably based on a bonding group containing at least an amide bondor an ester bond.

In addition, a probe to specifically trap the target material chemicallybonds with the saccharide in the magnetic particles for specifictrapping of this embodiment. Although not specifically limited, thesaccharide and the probe may bond via a chemical bond such as an —O—bond, an —S— bond, an —SO— bond, an —SO₂— bond, a —CO— bond, a —CO₂—bond, an —NR— bond (wherein R is an alkyl group or H), an —N⁺R²R³— bond(wherein R² and R³ are individually an alkyl group or H), an —NHCO—bond, or a —PO₂— bond. The saccharide and the probe can be chemicallybonded by, for example, chemically reacting a functional group in thesaccharide with a functional group in the probe.

The functional group in the saccharide and the functional group in theprobe are not specifically limited. As examples, groups such as ahydroxyl group, an acyl group, a mercapto group, an amino group, anamino acyl group, a carbonyl group, a formyl group, a carboxyl group, anamide group, a sulfonic group, a phosphate group, an epoxy group, atosyl group, an azido group, a vinyl group, and an allyl group can begiven.

In this embodiment, the term “target material” refers to a target to betrapped by the magnetic particles for specific trapping according tothis embodiment. As an example of the target material, a biologicallyrelated material can be given. In the embodiment, the term“biologically-related material” refers to all materials relating tobiological bodies. As examples of the biologically-related material,materials contained in biological bodies, materials derived frommaterials contained in biological bodies, and materials which can beused in biological bodies can be given.

More specific examples of the biologically-related materials include,but are not limited to, proteins (e.g., enzymes, antibodies, andacceptors), peptides (e.g., glutathione, RGD peptides), nucleic acids(e.g., DNA and RNA), carbohydrates, lipids, and other cells andsubstances (e.g., various blood-originating substances and variousfloating cells containing various blood cells such as platelets,erythrocytes, and leukocytes).

When the probe is a protein, for example, the probe can be chemicallybonded to a saccharide by, for example, reacting a functional group inthe protein (for example, an amino group and a carboxyl group) with afunctional group in the saccharide (for example, a carboxyl group, ahydroxyl group, and an amino group). In this instance, the probe and thesaccharide can be bonded through an amide bond or an ester bond.

When the probe is a nucleic acid, for example, the probe can bechemically bonded to a saccharide by, for example, reacting a functionalgroup in the nucleic acid (for example, a phosphoric acid group) with afunctional group in the saccharide (for example, a hydroxyl group). Inthis instance, the probe and the saccharide can be bonded through aphosphodiester bond.

Although not particularly limited, the probe which can be used with theparticles for specific trapping includes, for example, a protein (forexample, an antibody, an antigen, an enzyme, an acceptor, and ahormone), a peptide, a nucleic acid (for example, DNA and RNA), aglycoside compound, and a synthetic chemical substance (for example, apharmaceutical candidate compound).

When the probe is an antibody (or an antigen), the target material maybe an antigen (or an antibody) which specifically bonds to the antibody(or the antigen).

When the probe is a nucleic acid, the target material may be a nucleicacid which specifically bonds to the nucleic acid. When the probe is anenzyme, an acceptor, or a hormone, the target material may be a chemicalcompound which specifically bonds to the enzyme, the acceptor, or thehormone.

Although the magnetic particles for specific trapping according to thisembodiment can be used as they are, in order to efficiently perform areaction with a compound, it is possible to use them dispersed in adispersion medium. As a dispersion medium, those given as the dispersionmedium in first embodiment may be used.

The particle diameter of the magnetic particles for specific trappingaccording to this embodiment is from 0.1 to 20 micrometers, preferablyfrom 0.3 to 17 micrometers, and more preferably from 0.5 to 10micrometers. If the particle diameter is less than 0.1 micrometer, ittakes a long time for separation using magnetism or the like, resultingin insufficient separation of the particles from a washing solvent suchas water. This makes it difficult to sufficiently remove materials otherthan the target material, giving rise to inadequate purification. On theother hand, if the particle diameter is more than 20 micrometers, theamount of the target material which can be trapped may be small due tothe surface area becoming smaller.

Each of the components of the magnetic particles for specific trappingof this embodiment are described below.

3-1-1. Magnetic Particles

The average particle diameter of the nuclear particles used in thisembodiment is preferably from 0.1 to 20 micrometers, more preferablyfrom 0.3 to 17 micrometers, and still more preferably from 0.5 to 10micrometers. If the particle diameter of the magnetic particles is lessthan 0.1 micrometer, it may take a long time for separation andpurification using magnetism; and if more than 20 micrometers, theamount of trapped target material may be small due to the surface areabecoming smaller.

As mentioned above, when the magnetic particles for specific trapping ofthis embodiment are dispersed in a solvent, the nonspecific adsorptionof the target materials increases if the magnetic particles aredissolved in the dispersion medium or the magnetic particles are swollenby the solvent. For this reason, it is desirable that the magneticparticles are not dissolved in a solvent.

Although the internal composition of the magnetic particles used in thisembodiment may be homogeneous, most magnetic materials making up thehomogeneous magnetic particles with a particle diameter in theabove-mentioned preferable range are paramagnetic. If repeatedlyseparated and refined by magnetism, the magnetic particles may losetheir capability of being dispersed in dispersion media. For thisreason, it is preferable that the magnetic particles of this embodimenthave a heterogeneous internal composition containing fine particles of amagnetic material exhibiting small residual magnetization. As the innerstructure of the magnetic particles having such a heterogeneous internalcomposition, (i) a structure in which the magnetic particles aredispersed in a continuous phase of a non-magnetic material such as apolymer, (ii) a structure consisting of a secondary aggregate of fineparticles of a magnetic material as a core and a non-magnetic materialsuch as a polymer layer as a shell, and (iii) a structure consisting ofa non-magnetic material such as a polymer (non-magnetic nuclearparticles) as a core and a magnetic material layer (secondary aggregatematerials of fine particles of a magnetic material) of supermagneticnanoparticles provided on the surface of the nuclear particles, and thelike can be given. As the polymer which can be used as the core,polymers described later as the polymer forming the magnetic particlesmay be used. The fine particles of a magnetic material in the abovestructures (i) to (iii) are preferably fine particles of at least one ofFe₂O₃ and Fe₃O₄.

In the case of the structure of (iii) above, in which the innerstructure consists of a core of a non-magnetic material such as apolymer (non-magnetic nuclear particles) and a shell of a magneticmaterial layer (a secondary aggregate of fine particles of a magneticmaterial), it is preferable that a polymer layer be further formed onthe magnetic material layer. In this instance, the polymer layer may beformed by polymerization on the surface of the mother particles whichcontain nuclear particles (core) and a magnetic material layer (shell)formed on the surface of the nuclear particles. The magnetic materiallayer (shell) may contain fine particles of a magnetic material whichcontain at least one of Fe₂O₃ and Fe₃O₄. As the polymer which can beused for the polymer layer, polymers described later as the polymerforming the magnetic particles may be used.

In the case of the above structure (iii), the magnetic material layermay be produced by, for example, mixing the non-magnetic materialnuclear particles with the fine particles of a magnetic material andcausing the fine particles of a magnetic material to be physicallyadsorbed on the surface of the non-magnetic material nuclear particles.In this embodiment, “physical adsorption” refers to adsorption notinvolving a chemical reaction. As the principle of “physicaladsorption”, hydrophobic/hydrophobic adsorption, molten bonding oradsorption, fusion bonding or adsorption, hydrogen bonding,Van-der-Waals bonding, and the like can be given, for example.

The magnetic particles of the structure (iii) above can be obtained by,for example, suspension polymerization of the above vinyl monomer orpolymer bulk shattering. For example, the magnetic particles can beobtained by the two-stage swelling polymerization method using seedparticles described in JP-UM-B-57-24369, the polymerization methoddescribed in J. Polym. Sci., Polymer Letter Ed., 21, 937 (1963), and themethods described in JP-A-61-215602, JP-A-61-215603, and JP-A-61-215604.

The magnetic particles of the structure (iii) above can also be preparedby a method utilizing hydrophobic/hydrophobic adsorption. For example, amethod selecting non-magnetic nuclear particles and fine particles of amagnetic material, each having a hydrophobic or hydrophobized surface,and dry-blending these non-magnetic nuclear particles and fine particlesof a magnetic material, and a method sufficiently dispersing thenon-magnetic nuclear particles and fine particles of a magnetic materialin a solvent (such as toluene or hexane) with good dispersibilitywithout damaging both particles, followed by vaporization of the solventwhile mixing can be given.

Alternatively, in the case of the above structure (iii), the magneticparticles may be produced by physically applying a strong external forceto cause the fine particles of a magnetic material to be adsorbed on thesurface of the non-magnetic material nuclear particles. As examples ofthe method for physically applying a strong force, a method using amortar, an automatic mortar, or a ball mill; a blade-pressuring typepowder compressing method; a method utilizing a mechanochemical effectsuch as a mechanofusion method; and a method using an impact in ahigh-speed air stream such as a jet mill, a hybridizer, or the like canbe given. In order to efficiently produce a firmly bound complex, astrong physical adsorption force is desirable. As the method, stirringusing a vessel equipped with a stirrer at a peripheral velocity ofstirring blades of preferably 15 m/sec or more, more preferably 30 m/secor more, and still more preferably from 40 to 150 m/sec can be given. Ifthe stirring blade peripheral velocity is less than 15 m/sec, sufficientenergy for causing fine particles of a magnetic material to be adsorbedon the surface of non-magnetic nuclear particles may not be obtained.Although there are no specific limitations to the upper limit of theperipheral speed of the stirring blades, the upper limit of theperipheral speed is determined according to the apparatus to be used,energy efficiency, and the like.

As the polymer used for forming the magnetic particles of thisembodiment, those mentioned in connection with the organic polymerparticles of the first embodiment can be given.

3-1-2. Saccharide

As the saccharide used for forming the magnetic particles for specifictrapping of this embodiment, those mentioned in the first embodiment canbe given.

3-2. Process for Producing Magnetic Particles for Specific Trapping

The process for producing the magnetic particles for specific trappingaccording to this embodiment comprises chemically bonding magneticparticles with a diameter of 0.1 to 20 micrometers with a saccharide andchemically bonding a probe for specifically trapping the target materialwith a saccharide. The surface of the magnetic particles can be coveredwith a saccharide by chemically bonding the magnetic particles with thesaccharide.

In this embodiment, a general chemical reaction may be used forchemically bonding the magnetic particles with the saccharide withoutany particular limitations. For example, the magnetic particles used forproducing the magnetic particles for specific trapping according to thisembodiment may have two or more functional groups (first functionalgroups) on the surface. The first functional group may be a functionalgroup introduced when the particle shape of magnetic particles is formedor a functional group obtained by converting the functional group afterthe particle shape of magnetic particles has been formed. The conversionof functional groups may be carried out two or more times. Although notparticularly limited, when the functional group introduced when formingthe particle shape of the magnetic particles is an epoxy group, an aminogroup produced by reacting the epoxy group with a large excess amount ofammonia or an appropriate diamine compound may be the first functionalgroup, or when the functional group introduced when forming the particleshape of the magnetic particles is a hydroxyl group, for example, anamino group produced by converting the hydroxyl group into a tosyl groupand reacting the tosyl group with a large excess amount of anappropriate diamine compound may be the first functional group. Forexample, in the magnetic particles Am-6 and Am-7 respectively obtainedin the later-described Experimental Examples 8 and 9, the firstfunctional group can be the amino group.

The saccharide used for producing the magnetic particles for specifictrapping according to this embodiment may have two or more functionalgroups (second functional groups) in the molecule.

As the functional group which can be used as the first functional groupand/or the second functional group, a carboxyl group, a hydroxyl group,an epoxy group, an amino group, a mercapto group, a vinyl group, anallyl group, an acrylic group, a methacryl group, a tosyl group, anazido group, and the like can be given. The first functional group andthe second functional group are reactive with each other. Although notparticularly limited, when the first functional group is an epoxy group,for example, the second functional group may be an amino group, or whenthe first functional group is an amino group, for example, the secondfunctional group may be a carboxyl group.

It is possible to chemically bond the magnetic particles with thesaccharide by reacting the first functional group and the secondfunctional group, whereby the magnetic particles for specific trappingaccording to this embodiment can be obtained.

In this embodiment, a general chemical reaction may be used forchemically bonding the magnetic particles to the saccharide without anyparticular limitations. The probe and the saccharide can be chemicallybonded by, for example, chemically reacting a functional group in theprobe with a functional group in the saccharide. The functional groupcontained in the probe and the functional group contained in thesaccharide are the groups mentioned above.

After preparation according to the above-described process, the magneticparticles for specific trapping of this embodiment can be used ascarrier particles after adjusting the pH and washing the surface bypurification processing such as dialysis, ultrafiltration, andcentrifugation, as required.

4. Examples

The invention will now be described in more detail by way of examples,which should not be construed as limiting the invention. In theExamples, “%” and “part” are indicated on the weight basis.

4-1. Example 1 4-1-1. Evaluation Method 4-1-1A. Evaluation 1 ofNonspecific Adsorption (Protein Adsorption) 4-1-1A-1. Pre-Washing Step

Carrier polymer particles prepared in the later-described ExperimentalExamples and Comparative Examples were diluted with and dispersed inpurified water to obtain dispersion liquids, each having a particleconcentration of 1 wt %. 500 microliters of the dispersion liquid wasput into a microcentrifuge tube (“Safe-Lock Tube” manufactured byEppendorf AG) and centrifuged (15,000 rpm, 15° C., 10 minutes) using acentrifugal separator (“MX-150” manufactured by Tomy Seiki Co.) toremove the supernatant liquid. 500 microliters of a PBS(−) buffersolution was added to the tube which contained the precipitate, and themixture was vibrated by a touch mixer to disperse the particles.

4-1-1A-2. Protein Adsorption Reaction Step

Then, 500 microliters of a PBS(−) solution of 1 wt % BSA (bovine serumalbumin) was added to the tube and the mixture was vibrated by a touchmixer to disperse the particles in the solution, followed by mixing byrotation and inversion for two hours at room temperature.

4-1-1A-3. Washing Step

After centrifugal separation, the supernatant liquid was removed. 1 mlof 10 mM HEPES was added to the tube and the particles were dispersed byvibration using a touch mixer. After repeating the same procedure twice,the content was transferred to another microcentrifuge tube to performcentrifugal separation, and the supernatant liquid was removed.

4-1-1A-4. Detaching Step

After the addition of 50 microliters of a 0.5% aqueous solution of SDS(sodium dodecylsulfate), the mixture was gently vibrated by a touchmixer to disperse the particles. After allowing the mixture to stand for10 minutes, centrifugal separation was performed and 20 microliters ofthe supernatant liquid was collected.

4-1-1A-5. Sampling Step

2-mercaptoethanol was dissolved in a premix sample buffer solutionmanufactured by Bio-Rad Laboratories, Inc. to a concentration of 2 wt %(this solution is hereinafter referred to as “sample buffer”). 20microliters of the solution was collected in the microcentrifuge tube.20 microliters of the supernatant liquid collected in the above step wasmixed and heated at 100° C. for five minutes in a tube heater.

As controls, a 1 wt % BSA solution in PBS(−) was diluted with a 2% SDSsolution to 5,000 fold, 10,000 fold, and 20,000 fold. 20 microliters ofeach of the diluted solutions was mixed with 20 microliters of thesample buffer and heated in a tube heated at 100° C. for five minutes.The resulting solutions are called “reference diluted BSA”.

4-1-1A-6. Electrophoresis (SDS-PAGE)

The reference diluted BSA was applied to a vertical electrophoresissystem (“Mini-PROTEAN3” manufactured by Bio-Rad Laboratories, Inc.) inan amount of 20 microliters per one lane of the gel to performelectrophoresis using a precast polyacrylamide gel (“Ready Gel J” (15%)manufactured by Bio-Rad Laboratories, Inc.) and a premix electrophoresisbuffer solution manufactured by Bio-Rad Laboratories, Inc. The gel wasstained by a standard staining method using “Silver Stain Plus Kit”manufactured by Bio-Rad Laboratories, Inc. An image was produced byscanning the stained gel using a densitometer “GS-700” manufactured byBio-Rad Laboratories, Inc. and the product of the concentration and thearea of the BSA band in the gel was determined using an analysissoftware “Multi-Analyst”.

Since the weight of BSA which flows per one lane of the gel is known inthe reference dilution BSA, a calibration curve was drawn from theproduct of the band concentration and the area, and the amount of BSAdetached from the particles was converted on a weight basis based on thecalibration curve. The resulting weight corresponded to the amount ofBSA which had been adsorbed per 1 mg of the particles.

4-1-1B. Particle Diameter

The diameter of the particles with a diameter of 1 micrometer or morewas measured using a laser diffraction particle size distributionanalyzer (“SALD-200V” manufactured by Shimadzu Corp.) and the diameterof the particles with a diameter of less than 1 micrometer was measuredusing a particle size distribution analyzer based on a laser dispersiondiffraction method (“LS 13 320” manufactured by Beckmann Coulter).

4-1-1C. Infrared Absorption Spectrum

The infrared absorption spectrum was measured by a KBr method using aFourier-transform infrared spectrophotometer (“JIR-5500” manufactured byJEOL Ltd.).

4-1-2. Synthesis Examples 4-1-2A. Synthesis Example 1 Synthesis ofOrganic Polymer Particles A-1

The organic polymer particles A-1 were prepared by a two-step swellingpolymerization method using seed particles.

Using polystyrene particles with a particle diameter of 0.98 micrometersobtained by soap-free polymerization as seed particles, a waterdispersion (solid content: 5.0 g) was prepared by dispersing thesepolystyrene particles in 500 g of water in a nitrogen atmosphere.According to the two step swelling polymerization method (based on themethod described in JP-B-57-24369), an organic solvent (0.1 g of“Shellsol TK”) was added to the seed particles as a first step andmonomers (70 g of MMA (methyl methacrylate), 10 g of TMP(trimethylolpropane trimethacrylate), and 20 g of GMA (glycidylmethacrylate)) were added as a second step to cause them to be adsorbed.Then, 2 g of AIBN (azobisisobutyronitrile) was added and the mixture wasslowly stirred at 75° C. for 24 hours. The reaction solution was cooledand filtered through a 500 mesh wire gauze to confirm that 99% of theproduct passed through the wire gauze. The polymerization stability wasgood. The polymerization yield was 99%. The particle diameter of theresulting organic polymer particles A-1 was 2.71 micrometers, thecoefficient of variation of the particle diameter was 2%, and theparticles were monodisperse particles.

4-1-2B. Synthesis Example 2 Synthesis of Organic Polymer Particles A-2

Organic polymer particles A-2 with a particle diameter of 2.64micrometers and a coefficient of variation of 2% were obtained in thesame manner as in Synthetic Example 1, except for using 50 g of MMA, 10g of TMP, and 40 g of GMA as monomers.

4-1-2C. Synthesis Example 3 Synthesis of Organic Polymer Particles A-3

Organic polymer particles A-3 with a particle diameter of 2.61micrometers and a coefficient of variation of 2.1% were obtained in thesame manner as in Synthetic Example 1, except for using 30 g of MMA, 10g of TMP, and 60 g of GMA as monomers.

4-1-2D. Synthesis Example 4 Synthesis of Organic Polymer Particles A-4

Organic polymer particles A-4 with a particle diameter of 7.05micrometers and a coefficient of variation of 2.3% were obtained in thesame manner as in Synthetic Example 3, except for using polystyreneparticles with a particle diameter of 2.6 micrometers as seed particles.

4-1-2E. Synthesis Example 5 Synthesis of Organic Polymer Particles A-5

Organic polymer particles A-5 with a particle diameter of 2.58micrometers and a coefficient of variation of 2.3% were obtained in thesame manner as in Synthetic Example 1, except for using 10 g of TMP and90 g of GMA as monomers.

4-1-2F. Synthesis Example 6 Synthesis of Saccharide CMC-1

Diluted hydrochloric acid was added to an aqueous solution ofcarboxymethylcellulose sodium salt (“APP-84” manufactured by NipponPaper Chemicals Co., Ltd., a compound having an average molecular weightof 17,000 and an average of 0.7 carboxyl groups per one glucose unit)until the solution has a pH of 2 or less. The resulting solution wasdialyzed and concentrated to obtain a 2.5% aqueous solution ofcarboxymethylcellulose CMC-1.

4-1-2G Synthesis Example 7 Synthesis of Saccharide CMD-1

0.72 g of sodium hydroxide and 1.04 g of bromoacetic acid were added to2.5 g of a 10 wt % aqueous solution of Dextran T500 (average molecularweight: 500,000) manufactured by Pharmacia AB, and the mixture wasstirred for several minutes until homogenized. The solution wasmaintained at 40° C. for 60 hours and then cooled with ice. After theaddition of diluted hydrochloric acid to make the pH 2 or less, thesolution was dialyzed and freeze-dried to obtain carboxymethyldextranCMD-1. Carboxylic acid contained in CMD-1 was measured by titration tofind that CMD-1 contained an average of 0.4 carboxylic acid groups perone glucose unit.

4-1-3. Experimental Example 1

The polymer particles isolated from the dispersion liquid of organicpolymer particles A-1 by centrifugation were washed by dispersing inacetone, followed by centrifugation. This washing procedure was repeatedthree times. The resulting particles were dried. 0.50 g of the particleswas put into a 100 ml flask and 25 g of ethylenediamine was added. Theparticles were irradiated with indirect ultrasonic radiation for 10minutes and dispersed. The dispersion liquid was stirred at 50° C. in anitrogen atmosphere for six hours, followed by isolation of theparticles by centrifugal separation. The particles were washed twicewith methanol and three times with a 3:1 (by volume) mixture of waterand methanol, and dried to obtain 0.54 g of organic polymer particlesAm-1 as a white powder.

The weight of the organic polymer particles Am-1 was larger than theweight of the organic polymer particles A-1. Comparison of the infraredabsorption spectrum of the organic polymer particles Am-1 (afterethylenediamine treatment) with the infrared absorption spectrum of theorganic polymer particles A-1 (before ethylenediamine treatment)indicates that in the infrared absorption spectrum of the organicpolymer particles Am-1, a peak originating from an epoxy group, whichwas observed around 900 cm⁻¹ of the infrared absorption spectrum of theorganic polymer particles A-1, disappeared and, instead, peaks typicalto a primary amine appeared around 3,300 cm⁻¹ and 3,500 cm⁻¹. Theorganic polymer particles Am-1 were thus confirmed to have an aminogroup introduced into the organic polymer particles A-1. That is, in theorganic polymer particles Am-1, the first functional group 13 is anamino group.

30.6 mg of the organic polymer particles Am-1 was added to 1.2 g of a2.5% aqueous solution of CMC-1 which was obtained in Synthetic Example6. The mixture was irradiated with indirect supersonic waves for 30minutes to disperse the polymer particles in the solution. Next, thedispersion liquid was cooled with ice, and 0.30 g of a 10 wt % aqueoussolution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloridewas added. The mixture was stirred under ice cooling for 12 hours. Theparticles were isolated by centrifugal separation, dispersed in purifiedwater, isolated by centrifugal separation, and washed. This procedurewas repeated 10 times, followed by drying to obtain 33.6 mg of carrierpolymer particles P-1.

In addition to the peaks originating from the organic polymer particlesAm-1 before the reaction, peaks originating from carboxymethylcellulosewere observed around 3,400 cm⁻¹ and 1,600 cm⁻¹ in the infraredabsorption spectrum of the carrier polymer particles P-1. The carrierpolymer particles P-1 were thus confirmed to have a saccharide(carboxymethylcellulose) bonded to the organic polymer particles Am-1.

The nonspecific protein adsorption of the carrier polymer particles P-1was measured according the above-described method to confirm that thevalue was very low (0.08 ng/mg).

4-1-4. Experimental Example 2

0.57 g of organic polymer particles Am-2 were obtained in the samemanner as in Experimental Example 1, except for using a dispersionliquid of organic polymer particles A-2. Next, 34.0 mg ofcarboxymethylcellulose-bonded particles (carrier polymer particles) P-2was obtained in the same manner as in Experimental Example 1, except forusing the organic polymer particles Am-2 (29.6 mg) and a 2.5% aqueoussolution of CMC-1 (1.2 g).

The nonspecific protein adsorption of the carrier polymer particles P-2was measured according the above-described evaluation method to confirmthat the value was very low (0.05 ng/mg).

4-1-5. Experimental Example 3

0.61 g of organic polymer particles Am-3 was obtained in the same manneras in Experimental Example 1, except for using a dispersion liquid ofthe organic polymer particles A-3. 36.2 mg ofcarboxymethylcellulose-bonded particles (carrier polymer particles) P-3was obtained in the same manner as in Experimental Example 1, except forusing the organic polymer particles Am-3 (29.9 mg) and a 2.5% aqueoussolution of CMC-1 (1.2 g).

The nonspecific protein adsorption of the carrier polymer particles P-3was measured according the above-described method to confirm that thevalue was very low (0.02 ng/mg).

4-1-6. Experimental Example 4

150 mg of CMD-1 which was obtained in Synthetic Example 7 was dissolvedin 6 g of purified water. 150.5 mg of the organic polymer particles Am-1which were obtained in Experimental Example 1 was added to the solution,and the mixture was irradiated with indirect supersonic waves for 30minutes to disperse the particles in the solution. Next, the dispersionliquid was cooled with ice, and 1.40 g of a 5 wt % aqueous solution of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added.The mixture was stirred under ice cooling for 12 hours. The particleswere isolated by centrifugal separation, dispersed in purified water,isolated by centrifugal separation, and washed. This procedure wasrepeated 10 times to obtain 156.2 mg of carrier polymer particles P-4.

In addition to the peaks originating from the organic polymer particlesAm-1 before the reaction, peaks originating from carboxymethyldextranwere observed around 3,400 cm⁻¹ and 1,600 cm⁻¹ in the infraredabsorption spectrum of the carrier polymer particles P-4. The carrierpolymer particles P-4 were thus confirmed to have a saccharide(carboxymethyldextran) bonded to the organic polymer particles Am-1.

The nonspecific protein adsorption of the carrier polymer particles P-4was measured according the above-described evaluation method to confirmthat the value was very low (0.07 ng/mg).

4-1-7. Experimental Example 5

0.62 g of organic polymer particles Am-4 was obtained in the same manneras in Experimental Example 1, except for using a dispersion liquid ofthe organic polymer particles A-4. 36.1 mg ofcarboxymethylcellulose-bonded particles (carrier polymer particles) P-5was obtained in the same manner as in Experimental Example 1, except forusing the organic polymer particles Am-4 (29.9 mg) and a 2.5% aqueoussolution of CMC-1 (1.2 g).

The nonspecific protein adsorption of the carrier polymer particles P-5was measured according the above-described evaluation method to confirmthat the value was very low (0.05 ng/mg).

4-1-8. Experimental Example 6

The polymer particles isolated from the dispersion liquid of organicpolymer particles A-5 by centrifugation were washed by dispersing inacetone, followed by centrifugation. This washing procedure was repeatedthree times. The resulting particles were dried.

0.50 g of the particles was put into a 100 ml flask and 46.5 g ofdimethylsulfoxide was added. The mixture was irradiated with indirectultrasonic radiation for 10 minutes and, after the addition of 3.5 g ofa 10 wt % ethylenediamine solution (solvent: dimethylsulfoxide), themixture was stirred at 50° C. in a nitrogen atmosphere for four hours,followed by isolation of the particles by centrifugal separation. Theparticles were washed twice with methanol and three times with a 3:1 (byvolume) mixture of water and methanol, and dried to obtain 0.55 g oforganic polymer particles Am-5 as a white powder.

The weight of the organic polymer particles Am-5 was larger than theweight of the organic polymer particles A-5. Comparison of the infraredabsorption spectrum of the organic polymer particles Am-5 (afterethylenediamine treatment) with the infrared absorption spectrum of theorganic polymer particles A-5 (before ethylenediamine treatment)indicates that in the infrared absorption spectrum of the organicpolymer particles Am-5, a peak originating from an epoxy group, whichwas observed around 900 cm⁻¹ of the infrared absorption spectrum of theorganic polymer particles A-5, disappeared and, instead, peaks typicalto a primary amine appeared around 3,300 cm⁻¹ and 3,500 cm⁻¹. Based onthe results, the organic polymer particles Am-5 were confirmed to havean amino group introduced into the organic polymer particles A-5. Basedon the change in the peak intensity originating from the epoxy groupbefore and after the treatment with ethylenediamine, the reaction rateof the epoxy group which exists in the organic polymer particles A-5 waspresumed to be about 30%.

100 mg of the organic polymer particles Am-5 was added to a mixture of15 g of 1 wt % sulfuric acid and 1.5 g of acetone. The mixture wasirradiated with indirect ultrasonic radiation for 10 minutes anddispersed. The dispersion liquid was stirred at 50° C. for nine hours,followed by isolation of the particles by centrifugal separation.

The particles were washed three times with water, once with a 0.01 Nsodium hydroxide aqueous solution, and five times with water, and driedto obtain 102 mg of particles.

Comparison of the infrared absorption spectrum of the organic polymerparticles Am-5 before reaction with the infrared absorption spectrum ofthe particles obtained here (after the treatment with sulfuric acid)indicates that in the infrared absorption spectrum of the organicpolymer particles Am-5, intensity of a peak originating from an epoxygroup, which was observed around 900 cm⁻¹ of the infrared absorptionspectrum of the organic polymer particles Am-5, disappeared and,instead, a peak originating from a hydroxyl group around 3,500 cm⁻¹ wasintensified. Based on the results, it was confirmed that the epoxy groupin the organic polymer particles Am-5 has been hydrolyzed.

33.1 mg of carboxymethylcellulose-bonded particles (carrier polymerparticles) P-6 was obtained in the same manner as in ExperimentalExample 1, except for using 30.3 mg of the particles obtained above.

The nonspecific protein adsorption of the carrier polymer particles P-6was measured according the above-described method to confirm that thevalue was less than the detectable limit (0.01 ng/mg).

4-1-9. Experimental Example 7

Sodium salt of carboxymethylcellulose (“APP-84” manufactured by NipponPaper Industries Chemical, Inc.) was purified by dialyzing an aqueoussolution, followed by freeze-drying. An experiment was carried out inthe same manner as in Experimental Example 1, except for using thepurified APP-84 (33 mg) and the organic polymer particles Am-7 (29.8mg). Next, the particles were isolated by centrifugal separation,dispersed in purified water, and washed. This procedure was carried outfive times, a procedure of dispersing the particles in a 0.01 Nhydrochloric acid solution was carried out three times, and a procedureof dispersing in purified water, followed by centrifugal separation wascarried out five times. The resulting particles were dried to obtain33.7 mg of carboxymethylcellulose-bonded particles (carrier polymerparticles) P-7.

The nonspecific protein adsorption of the carrier polymer particles P-7was measured according the above-described evaluation method to confirmthat the value was very low (0.05 ng/mg).

4-1-10. Comparative Example 1

The nonspecific protein adsorption of the organic polymer particles A-1was measured according the above-described method to confirm that thevalue was high (1.3 ng/mg).

4-1-11. Comparative Example 2

Commercially available standard polystyrene particles (“STADEX SC200S”manufactured by JSR Corporation) was sufficiently washed with purifiedwater and their nonspecific protein adsorption was measured accordingthe above-described method to confirm that the value was very high (20ng/mg).

4-1-12. Comparative Example 3

Particles of which the surface was covered with polyethylene glycol wereobtained in the same manner as in Experimental Example 1, except forusing a 2.5% aqueous solution of polyethylene glycol with both terminalsmodified with carboxylic acid (the number of average repetition ofethylene oxide unit: 10) instead of a 2.5% aqueous solution of CMC-1.The nonspecific protein adsorption of P-8 was measured according theabove-described method to confirm that the value was 0.3 ng/mg.

4-2. Example 2 4-2-1. Method of Evaluation of Properties 4-2-1A.Particle Diameter

The diameter of the particles with a diameter of 1 micrometer or morewas measured using a laser diffraction particle size distributionanalyzer (“SALD-200V” manufactured by Shimadzu Corp.) and the diameterof the particles with a diameter of less than 1 micrometer was measuredusing a particle size distribution analyzer based on a laser dispersiondiffraction method (“LS 13 320” manufactured by Beckmann Coulter).

4-2-1B. Infrared Absorption Spectrum

The infrared absorption spectrum was measured by a KBr method using aFourier-transform infrared spectrophotometer (“JIR-5500” manufactured byJEOL Ltd.).

]

4-2-2. Synthesis Examples 4-2-2A. Synthesis Example 8 Synthesis ofMagnetic Particles A-6

Referring to the polymerization method described in JP-A-7-238105,styrene/divinylbenzene (96/4) copolymer particles (average particlediameter: 1.5 micrometers) were prepared. After polymerization, theparticles were separated by centrifugation, washed with water, dried,and ground. The ground particles were used as core particles (a-1)(preparation of core particles).

Next, ferrite-type fine particles of a magnetic material (averageprimary particle diameter: 0.02 micrometers) with a hydrophobizedsurface were prepared by adding acetone to an oily magnetic fluid (“EXPseries” manufactured by Ferrotec Corp.) to obtain a precipitate of theparticles and drying the precipitate.

Then, 15 g of the above core particles (a-1) and 15 g of thehydrophobized fine particles of a magnetic material were thoroughlymixed in a mixer. The mixture was processed by a hybridization system(“NHS-0 type” manufactured by Nara Machinery Co., Ltd.) at a peripheralblade (stirring blades) speed of 100 m/sec (16,200 rpm) for five minutesto obtain particles (1) with a magnetic material layer of fine particlesof a magnetic material (M−1) with a number average particle diameter of2.0 micrometers on the surface (preparation of magnetic material layer).

A 500 ml separable flask was charged with 375 g of an aqueous solutioncontaining 0.25 wt % of sodium dodecylbenzenesulfonate and 0.25 wt % ofa nonionic emulsifying agent (“Emulgen 150” manufactured by Kao Corp.),followed by the addition of 15 g of the above particles (1) having amagnetic material layer on the surface. After dispersion using ahomogenizer, the resulting dispersion liquid was heated to 60° C. Next,a pre-emulsion, prepared by dispersing 27 g of MMA (methylmethacrylate), 3 g of TMP (trimethylolpropane trimethacrylate), and 0.6g of di(3,5,5-trimethylhexanoyl) peroxide (“Peroyl 355” manufactured byNOF Corp.) in 150 g of an aqueous solution containing 0.25 wt % ofsodium dodecylbenzenesulfonate and 0.25 wt % of a nonionic emulsifyingagent (“Emulgen 150” manufactured by Kao Corp.), was dripped into theabove 500 ml separable flask controlled at 60° C. over one and a halfhours (a first stage polymerization for polymer layer formation).

After completing the dripping, the mixture was maintained at 60° C.while stirring for one hour. Next, a pre-emulsion, prepared bydispersing 7.5 g of MMA, 6 g of GMA (glycidyl methacrylate), 1.5 g ofTMP, and 0.3 g of di(3,5,5-trimethylhexanoyl) peroxide (“Peroyl 355”manufactured by NOF Corp.) in 75 g of an aqueous solution containing0.25 wt % of sodium dodecylbenzenesulfonate and 0.25 wt % of a nonionicemulsifying agent (“Emulgen 150” manufactured by Kao Corp.), was drippedto the above 500 ml separable flask controlled at 60° C. over one and ahalf hours (a second stage polymerization for polymer layer formation).After heating to 75° C., the polymerization was continued for a furthertwo hours before completing the reaction. The resulting water dispersionof polymer-covered magnetic particles was purified by magnetism andgravity precipitation to obtain a water dispersion of magnetic particlesA-6 with a solid component concentration of 1%. The number averageparticle diameter of the magnetic particles A-6 was 2.9 micrometers.

4-2-2B. Synthesis Example 9 Synthesis of Magnetic Particles A-7

A 500 ml separable flask was charged with 225 g of a 0.5 wt % sodiumdodecylbenzenesulfonate aqueous solution. 9 g of the particles (1)having a magnetic material layer were added and dispersed using ahomogenizer, and the resulting dispersion liquid was heated to 60° C. Apre-emulsion, prepared by dispersing 16.2 g of MMA, 1.8 g of TMP, and0.36 g of di(3,5,5-trimethylhexanoyl)peroxide (“Peroyl 355” manufacturedby NOF Corp.) in 90 g of an aqueous solution containing 0.5 wt % ofsodium dodecylbenzenesulfonate was dripped into the above 500 mlseparable flask controlled at 60° C. over one and a half hours (a firststage polymerization for polymer layer formation).

After completion of dripping, the mixture was maintained at 60° C. forone hour while stirring. A pre-emulsion, prepared by dispersing 8.1 g ofGMA, 0.9 g of TMP, and 0.18 g of di(3,5,5-trimethylhexanoyl)peroxide(“Peroyl 355” manufactured by NOF Corp.) in 45 g of an aqueous solutioncontaining 0.5 wt % of sodium dodecylbenzenesulfonate was dripped intothe above 500 ml separable flask controlled at 60° C. over one and ahalf hours (a second stage polymerization for polymer layer formation).After heating to 75° C., the polymerization was continued for two hoursbefore completing the reaction. The resulting water dispersion ofpolymer-covered magnetic particles was purified by magnetism and gravityprecipitation to obtain a water dispersion of magnetic particles A-7with a solid component concentration of 1%. The number average particlediameter of the magnetic particles A-7 was 2.6 micrometers.

4-2-2C. Synthesis Example 10 Synthesis of Saccharide CMC-1

Diluted hydrochloric acid was added to an aqueous solution ofcarboxymethylcellulose sodium salt (“APP-84” manufactured by NipponPaper Chemicals Co., Ltd., a compound having an average molecular weightof 17,000 and an average of 0.7 carboxyl groups per one glucose unit)until the solution has a pH of 2 or less. The resulting solution wasdialyzed and concentrated to obtain a 1% aqueous solution of acarboxymethylcellulose CMC-1.

4-2-2D. Synthesis Example 11 Synthesis of Saccharide CMD-1

0.72 g of sodium hydroxide and 1.04 g of bromoacetic acid were added to2.5 g of a 10 wt % aqueous solution of Dextran T500 (average molecularweight: 500,000) manufactured by Pharmacia AB, and the mixture wasstirred for several minutes to homogenize. The solution was maintainedat 40° C. for 60 hours and then cooled with ice. After the addition ofdiluted hydrochloric acid to make the pH 2 or less, the solution wasdialyzed and freeze-dried to obtain a carboxymethyldextran CMD-1.Carboxylic acid contained in CMD-1 was measured by titration to findthat CMD-1 contained an average of 0.4 carboxylic acid groups per oneglucose unit.

4-2-3. Experimental Example 8 4-2-3A. Preparation of Carrier PolymerParticles

Particles isolated from the water dispersion of magnetic particles A-6obtained in Synthetic Example 8 by centrifugal separation were dispersedin acetone. After repeating a procedure of separating the particles bymagnetism and washing five times, the particles were again dispersed inacetone and the supernatant liquid was removed by centrifugalseparation. The particles obtained were dried. 0.50 g of the particleswas put into a 100 ml flask and 25 g of ethylenediamine was added.Particles were irradiated with indirect ultrasonic radiation for 20minutes and dispersed. The dispersion liquid was stirred at 50° C. in anitrogen atmosphere for six hours, followed by isolation of theparticles by centrifugal separation. The particles were washed fivetimes with methanol and dried to obtain 0.49 g of aminated particlesAm-6 as a brown powder. Comparison of the infrared absorption spectrumof the aminated particles Am-6 (after ethylenediamine treatment) withthe infrared absorption spectrum of the magnetic particles A-6 (beforeethylenediamine treatment) indicates that peaks typical to a primaryamine appeared around 3,300 cm⁻¹ and 3,400 cm⁻¹ in the infraredabsorption spectrum of the aminated particles Am-6. The aminatedparticles Am-6 were thus confirmed to have an amino group introducedinto the magnetic organic polymer particles A-6.

150 mg of the aminated particles Am-6 was added to 3.75 g of a 1%aqueous solution of CMC-1 which was obtained in Synthetic Example 10.The dispersion liquid was irradiated with indirect supersonic waves for20 minutes while cooling with ice. Next, 25.05 mg of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added tothe dispersion liquid and the mixture was stirred under ice cooling for20 hours. The particles were isolated by magnetic separation anddispersed in purified water. A procedure of magnetic separation andwashing by dispersing in purified water was repeated five times. Theresulting particles were again dispersed in purified water andcentrifuged to remove the supernatant liquid, followed by drying toobtain 147 mg of carrier polymer particles P-9.

4-2-3B. Preparation of Probe-Bonded Polymer Particles

The carrier polymer particles obtained in Experimental Example 8 werediluted with and dispersed in purified water to obtain a waterdispersion with a particle concentration of 1 wt %. 500 microliters ofthe dispersion liquid was put into a microcentrifuge tube (“Safe-Locktube” manufactured by Eppendorf) and magnetically centrifuged using amagnetic stand (“Magical Trapper” manufactured by Toyobo Co., Ltd.) toremove the supernatant liquid. After washing three times with a 50 mMMES—NaOH buffer solution (pH 6, hereinafter referred to as “Buffer-1”),0.8 mg of N-hydroxysuccinic acid imide (NHS) and 0.88 mg of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) were added andstirred. Then, 0.05 mg of a protein (anti-alpha-fetoprotein antibody)which acts as a probe for specifically trapping the target material(alpha-fetoprotein, hereinafter referred to from time to time as “AFP”)was added and the mixture was stirred at room temperature for two hours.After the reaction, the particles were separated by magnetic separationand the supernatant liquid was removed. Then, 500 microliters of a PBS(−) buffer solution containing 1% ethanol amine was added and themixture was stirred at room temperature for two hours. Furthermore,after washing five times with a PBS (−) buffer solution, the particleswere dispersed in 500 microliters of PBS (−) buffer solution to obtain adispersion liquid of probe (antibody)-bonded particles.

4-2-3C. Evaluation of Specific Trapping Property

The specific trapping property of the probe-bonded particles obtained inthis Experimental Example was evaluated according to the followingmethod.

4-2-3C-1. Target Material (Protein) Adsorption Reaction Step

100 microliters of the above dispersion liquid of the probe-bondedparticles was sampled in a separate tube. Particles were magneticallyseparated to remove the supernatant liquid. 500 microliters of a humanblood serum solution containing 200 ng/ml of protein (humanalpha-fetoprotein (AFP)) which is the target material was added to theparticles. The mixture was vibrated by a touch mixer to disperse theparticles in the solution, followed by mixing by rotation and inversionfor two hours at room temperature.

4-2-3C-2. Washing Step

After magnetic separation, the supernatant liquid was removed. 1 ml of10 mM HEPES was added to the tube and the particles were dispersed usinga touch mixer. After further repeating the same procedure twice, thecontent was transferred to a new microcentrifuge tube to performmagnetic separation, and the supernatant liquid was removed.

4-2-3C-3. Detaching Step

After the addition of 50 microliters of a 0.5% aqueous solution of SDS(sodium dodecylsulfate), the mixture was gently vibrated to disperse theparticles. After allowing the mixture to stand for 10 minutes, magneticseparation was performed and 20 microliters of the supernatant liquidwas collected.

4-2-3C-4. Electrophoresis (SDS-PAGE)

2-mercaptoethanol was dissolved in a premix sample buffer solutionmanufactured by Bio-Rad Laboratories, Inc. to a concentration of 2 wt %(this solution is hereinafter referred to as “sample buffer”). 20microliters of the solution was collected in a microcentrifuge tube. 20microliters of the supernatant liquid collected in the above step wasmixed and heated at 100° C. for five minutes in a tube heater.

As controls, a 1 mg/ml AFP/BSA (−) solution was diluted with an SDSsolution to 100 fold, 200 fold, and 500 fold. 20 microliters of each ofthe diluted solutions were mixed with 20 microliters of the samplebuffer and heated by a block heater at 100° C. for five minutes. Theresulting solutions are called reference AFP dilutions.

The reference AFP dilutions were applied to a vertical electrophoresissystem (“Mini-PROTEAN3” manufactured by Bio-Rad Laboratories, Inc.) inan amount of 20 microliters per one lane to perform electrophoresisusing a precast polyacrylamide gel (“Ready Gel J” (15%) manufactured byBio-Rad Laboratories, Inc.) and a premix electrophoresis buffer solutionmanufactured by Bio-Rad Laboratories, Inc. The gel was stained by astandard staining method using “Silver Stain Plus Kit” manufactured byBio-Rad Laboratories, Inc. The stained gel was scanned using adensitometer “GS-700” manufactured by Bio-Rad Laboratories, Inc. toproduce an image. The product of the concentration and the area of theAFP band in the gel were determined using an analysis software“Multi-Analyst”.

Since the weight of AFP which flows per one lane of the gel is known inthe reference dilution AFP, a calibration curve was drawn from theproduct of the concentration and the area of the band, and the amount ofAFP detached from the particles was converted on a weight basis based onthe calibration curve. The resulting weight corresponded to the amountof AFP which had been adsorbed per 0.2 mg of the particles.

4-2-4. Experimental Example 9 4-2-4A. Preparation of Carrier PolymerParticles

0.48 g of aminated particles Am-7 were obtained in the same manner as inExperimental Example 8, except for using the magnetic particles A-7.CMD-1 (150 mg) which was obtained in Synthetic Example 11 was dissolvedin 6 g of purified water and 150.5 mg of the aminated particles Am-7 wasadded and dispersed in the solution by irradiation of indirectsupersonic waves for 20 minutes. Next, the dispersion liquid was cooledwith ice, and 1.40 g of a 5 wt % aqueous solution of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added.The mixture was stirred under ice cooling for 12 hours. The particleswere isolated by magnetic separation and dispersed in purified water. Aprocedure of magnetic separation and washing by dispersing in purifiedwater was repeated five times. The resulting particles were againdispersed in purified water and centrifuged to remove the supernatantliquid, followed by drying to obtain 147 mg of carrier polymer particlesP-10.

4-2-4B. Preparation of Probe-Bonded Polymer Particles and Evaluation ofSpecific Trapping Property

Probe (antibody)-bonded particles were obtained in the same manner as inExperimental Example 8 by using the carrier polymer particles P-10obtained in this Experimental Example. The specific trapping property ofthe probe-bonded particles was evaluated according to the same method asin Experimental Example 8.

4-2-5. Comparative Example 4

Probe-bonded particles were obtained in the same manner as inExperimental Example 8 by using magnetic particles “MAG2101”manufactured by JSR Corporation. The specific trapping property of theprobe-bonded particles was evaluated according to the same method as inExperimental Example 8. The surface of the magnetic particles used inthe Comparative Example 4 was not covered with a saccharide.

4-2-6. Evaluation Result of Specific Trapping Property

FIG. 3 is a photograph showing the evaluation results of the specifictrapping property (an electrophoresis pattern of proteins adsorbed onthe probe-bonded particles) obtained in Experimental Examples 8 and 9and Comparative Example 4.

In FIG. 3, lane 1 indicates proteins trapped by the probe-bondedparticles which were prepared by using the carrier polymer particles P-9of Experimental Example 8, lane 2 indicates proteins trapped by theprobe-bonded particles which were prepared by using the carrier polymerparticles P-10 of Experimental Example 9, lane 3 indicates proteinstrapped by the probe-bonded particles which were prepared by using themagnetic particles of Comparative Example 4, lane 4 indicates the targetmaterial (AFP) 20 ng which is a control, lane 5 indicates the targetmaterial (AFP) 50 ng which is a control, lane 6 indicates the targetmaterial (AFP) 100 ng which is a control, and lane 7 indicates amolecular weight marker.

It can be understood from FIG. 3 that by using the probe-bondedparticles which were prepared by using the polymer-bonded particles P-9of Experimental Example 8, only the target material (AFP) band wascollected from the serum in an amount of 11 ng per 0.2 mg of theparticles. By using the antibody-bonded particles which were prepared byusing the polymer-bonded particles P-10 of Experimental Example 9, onlythe target material (AFP) band was collected from the serum in an amountof 15 ng per 0.2 mg of the particles. On the other hand, although anumber of bands of blood serum proteins nonspecifically collected wereconfirmed in the particles of Comparative Example 4, it was difficult toconfirm the target AFP band.

The probe-bonded particles of Experimental Examples 8 and 9 were thusconfirmed to exhibit only small nonspecific protein adsorption. Based onthese results, it can be understood that since the surface of theparticles is covered with a saccharide and the probe to specificallytrap the target compound chemically bonds to the saccharide, theprobe-bonded particles of Experimental Examples 8 and 9 exhibit onlysmall nonspecific protein adsorption. On the other hand, the magneticparticles of Comparative Example 4 exhibited large nonspecificadsorption of proteins. It can thus be understood that the nonspecificadsorption is large if the surface of the particles is not covered witha saccharide.

4-3. Example 3 4-3-1. Evaluation Method 4-3-1A. Evaluation 1 ofNonspecific Adsorption (Evaluation of Nonspecific Adsorption ofProteins) 4-3-1A-1. Pre-Washing Step

The carrier polymer particles prepared in the later-describedExperimental Examples and Comparative Examples were diluted with anddispersed in purified water to obtain dispersion liquids, each having aparticle concentration of 1 wt %. 500 microliters of the dispersionliquid was put into a microcentrifuge tube (“Safe-Lock tube”manufactured by Eppendorf) and centrifuged (15,000 rpm, 15° C., 10minutes) using a centrifugal separator (“MX-150” manufactured by TomySeiki Co.) to remove the supernatant liquid. 500 microliters of a PBS(−)buffer solution was added to the tube which contained a precipitate, andthe mixture was vibrated by a touch mixer to disperse the particles.

4-3-1A-2. Protein Adsorption Reaction Step

Then, 500 microliters of a PBS(−) solution of 1 wt % BSA (bovine serumalbumin) was added to the tube and the mixture was vibrated by a touchmixer to disperse the particles in the solution, followed by mixing byrotation and inversion for two hours at room temperature.

]

4-3-1A-3. Washing Step

After centrifugal separation, the supernatant liquid was removed. 1 mlof 10 mM HEPES was added to the tube and the particles were dispersed byvibration using a touch mixer. After further repeating the sameprocedure twice, the content was transferred to another microcentrifugetube to perform centrifugal separation, and the supernatant liquid wasremoved.

4-3-1A-4. Detaching Step

After the addition of 50 microliters of a 0.5% aqueous solution of SDS(sodium dodecylsulfate), the mixture was gently vibrated by a touchmixer to disperse the particles. After allowing the mixture to stand for10 minutes, the centrifugal separation was performed and 20 microlitersof the supernatant liquid was collected.

4-3-1A-5. Sampling Step

2-mercaptoethanol was dissolved in a premix sample buffer solutionmanufactured by Bio-Rad Laboratories, Inc. to a concentration of 2 wt %(this solution is hereinafter referred to as “sample buffer”). 20microliters of the solution was collected in the microcentrifuge tube.20 microliters of the supernatant liquid collected in the above step wasmixed and heated at 100° C. for five minutes in a tube heater.

As controls, a 1 wt % BSA solution in PBS(−) was diluted with 2% SDS to5,000 fold, 10,000 fold, and 20,000 fold. 20 microliters of each of thediluted solutions was mixed with 20 microliters of the sample buffer andheated in a tube heated at 100° C. for five minutes. The resultingsolutions are called reference BSA dilutions.

4-3-1A-6. Electrophoresis (SDS-PAGE)

The reference AFP dilutions were applied to a vertical electrophoresissystem (“Mini-PROTEAN3” manufactured by Bio-Rad Laboratories, Inc.) inan amount of 20 microliters per one lane to perform electrophoresisusing a precast polyacrylamide gel (“Ready Gel J” (15%) manufactured byBio-Rad Laboratories, Inc.) and a premix electrophoresis buffer solutionmanufactured by Bio-Rad Laboratories, Inc. The gel was stained by astandard staining method using “Silver Stain Plus Kit” manufactured byBio-Rad Laboratories, Inc. The stained gel was scanned using adensitometer “GS-700” manufactured by Bio-Rad Laboratories, Inc. toproduce an image. The product of the concentration and the area of theBSA band in the gel were determined using an analysis software“Multi-Analyst”.

Since the weight of BSA which flows per one lane of the gel is known inthe dilution BSA for reference, a calibration curve was drawn from theproduct of the band concentration and the area, and the amount of BSAdetached from the particles was converted on a weight basis based on thecalibration curve. The resulting weight corresponded to the amount ofBSA which had been adsorbed per one mg of the particles.

4-3-1B. Evaluation of Specific Trapping Property 4-3-1B-1. Preparationof Probe-Bonded Polymer Particles

Carrier polymer particles prepared in the later-described ExperimentalExamples and Comparative Examples were diluted with and dispersed inpurified water to obtain dispersion liquids, each having a particleconcentration of 1 wt %. 500 microliters of the water dispersion was putinto a microcentrifuge tube and centrifuged to remove the supernatantliquid. After washing three times with a 50 mM MES—NaOH buffer solution(pH 6, hereinafter referred to as “Buffer-1”), 0.8 mg ofN-hydroxysuccinic acid imide (NHS) and 0.88 mg of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) were added andstirred. Then, 0.05 mg of a protein (anti-alpha-fetoprotein antibody)which acts as a probe for specifically catching the target material(alpha-fetoprotein, hereinafter referred to from time to time as “AFP”)was added and the mixture was stirred at room temperature for two hours.After the reaction, the particles were separated by centrifugalseparation and the supernatant liquid was removed. Then, 500 microlitersof a PBS (−) buffer solution containing 1% ethanol amine was added andthe mixture was stirred at room temperature for two hours. Furthermore,after washing five times with a PBS (−) buffer solution, the particleswere dispersed in 500 microliters of PBS (−) buffer solution to obtain adispersion liquid of probe (antibody)-bonded particles.

4-3-1B-2. Target Material (Protein) Adsorption Reaction Step

100 microliters of the above dispersion liquid of the probe-bondedparticles was sampled in a separate tube. The particles were separatedby centrifugation to remove the supernatant liquid. 500 microliters of ahuman blood serum solution containing the target protein (humanalpha-fetoprotein (AFP)) which is the target material was added to theparticles. The mixture was vibrated by a touch mixer to disperse theparticles in the solution, followed by mixing by rotation and inversionfor two hours at room temperature.

4-3-1B-3. Washing Step

After centrifugal separation, the supernatant liquid was removed. 1 mlof 10 mM HEPES was added to the tube and the particles were dispersedusing a touch mixer. After further repeating the same procedure twice,the content was transferred to another microcentrifuge tube to performcentrifugal separation, and the supernatant liquid was removed.

4-3-1B-4. Detaching Step

After the addition of 50 microliters of a 0.5% aqueous solution of SDS(sodium dodecylsulfate), the mixture was gently vibrated by a touchmixer to disperse the particles. After allowing the mixture to stand for10 minutes, the centrifugal separation was performed and 20 microlitersof a supernatant liquid was collected.

4-3-1B-5. Sampling Step

2-mercaptoethanol was dissolved in a premix sample buffer solutionmanufactured by Bio-Rad Laboratories, Inc. to a concentration of 2 wt %(this solution is hereinafter referred to as “sample buffer”). 20microliters of the solution was collected in the microcentrifuge tube.20 microliters of the supernatant liquid collected in the above step wasmixed and heated at 100° C. for five minutes in a tube heater.

As controls, a 1 mg/ml AFP/PBS (−) solution was diluted with an SDSsolution to 100 fold, 200 fold, and 500 fold. 20 microliters of each ofthe diluted solutions was mixed with 20 microliters of the sample bufferand heated by a block heater at 100° C. for five minutes. The resultingsolutions are called reference AFP dilutions.

4-3-1B-6. Electrophoresis (SDS-PAGE)

The electrophoresis was carried out in the same manner as in 4.1.1Fexcept for using AFP instead of BSA.

Since the weight of AFP which flows per one lane of the gel is known inthe reference dilution AFP, a calibration curve was drawn from theproduct of the concentration and the area of the band, and the amount ofAFP detached from the particles was converted on a weight basis based onthe calibration curve. The resulting weight corresponded to the amountof AFP which had been adsorbed per 0.2 mg of the particles.

4-3-1C. Particle Diameter

The diameter of the particles with a diameter of 1 micrometer or morewas measured using a laser diffraction particle size distributionanalyzer (“SALD-200V” manufactured by Shimadzu Corp.) and the diameterof the particles with a diameter of less than 1 micrometer was measuredusing a particle size distribution analyzer based on a laser dispersiondiffraction method (“LS 13 320” manufactured by Beckmann Coulter).

4-3-1D Infrared Absorption Spectrum

The infrared absorption spectrum was measured by a KBr method using aFourier-transform infrared spectrophotometer (“JIR-5500” manufactured byJEOL Ltd.).

4-3-2. Synthesis Examples 4-3-2A. Synthesis Example 12 Synthesis ofOrganic Polymer Particles A-8

The organic polymer particles A-8 were prepared by a two-step swellingpolymerization method using seed particles.

Using polystyrene particles with a particle diameter of 0.98 micrometersobtained by soap-free polymerization as seed particles, a waterdispersion (solid content: 5.0 g) was prepared by dispersing thesepolystyrene particles in 500 g of water in a nitrogen atmosphere.According to the two step swelling polymerization method (based on themethod described in JP-B-57-24369), an organic solvent (0.1 g of“Shellsol TK”) was added to the seed particles as a first step, andmonomers (10 g of TMP (trimethylolpropane trimethacrylate) and 90 g ofGMA (glycidyl methacrylate)) were added as a second step to cause themto be adsorbed. Then, 2 g of AIBN (azobisisobutyronitrile) was added andthe mixture was slowly stirred at 75° C. for 24 hours. The reactionsolution was cooled and filtered through a 500 mesh wire gauze toconfirm that 99% of the product passed through the wire gauze. Thepolymerization stability was good. The polymerization yield was 99%. Theparticle diameter of the resulting organic polymer particles A-8 was2.58 micrometers, the coefficient of variation of the particle diameterwas 2.3%, and the particles were monodisperse particles.

4-3-2B. Synthesis Example 13 Synthesis of Organic Polymer Particles A-9

Organic polymer particles A-9 with a particle diameter of 2.61micrometers and a coefficient of variation of 2.1% were obtained in thesame manner as in Synthetic Example 12, except for using 30 g of MMA, 10g of TMP, and 60 g of GMA as monomers.

4-3-2C. Synthesis Example 14 Synthesis of Saccharide CMC-1

Diluted hydrochloric acid was added to an aqueous solution ofcarboxymethylcellulose sodium salt (“APP-84” manufactured by NipponPaper Chemicals Co., Ltd., a compound having an average molecular weightof 17,000 and an average of 0.7 carboxyl groups per one glucose unit)until the solution has a pH of 2 or less. The resulting solution wasdialyzed and concentrated to obtain a 1% aqueous solution ofcarboxymethylcellulose CMC-1.

4-3-3. Experimental Example 10 4-3-3A. Preparation of Carrier PolymerParticles P-11

The polymer particles isolated from the dispersion liquid of organicpolymer particles A-8 by centrifugation were washed by dispersing inacetone, followed by centrifugation. This washing procedure was repeatedthree times. The resulting particles were dried. 0.50 g of the particleswas put into a 200 ml flask and 5 g of acetone and 75 g of 1% sulfuricacid were added. Particles were irradiated with indirect ultrasonicradiation for 20 minutes and dispersed. The dispersion liquid was heatedat 60° C. for two hours while stirring, followed by isolation of theparticles by centrifugal separation. The particles were washed threetimes with water and dried to obtain 0.51 g of organic polymer particlesHy-1 as a white powder.

The weight of the organic polymer particles Hy-1 was larger than theweight of the organic polymer particles A-8. Comparison of the infraredabsorption spectrum of the organic polymer particles Hy-1 (aftersulfuric acid treatment) with the infrared absorption spectrum of theorganic polymer particles A-8 (before sulfuric acid treatment) indicatesthat in the infrared absorption spectrum of the organic polymerparticles Hy-1, a peak originating from an epoxy group, which wasobserved around 900 cm⁻¹ of the infrared absorption spectrum of theorganic polymer particles A-8, was weak and, instead, a broad peak dueto a hydroxyl group was observed around 3,500 cm⁻¹. Based on the aboveresults, the organic polymer particles Hy-1 were confirmed to have beenobtained by a partial hydrolysis of epoxy groups in the organic polymerparticles A-8 and introduction of hydroxyl groups.

0.50 g of the organic polymer particles Hy-1 was put into a 100 ml flaskand 25 g of ethylenediamine was added. Particles were irradiated withindirect ultrasonic radiation for 10 minutes and dispersed. Thedispersion liquid was stirred at 50° C. in a nitrogen atmosphere for sixhours, followed by isolation of the particles by centrifugal separation.The particles were washed four times with methanol and dried to obtain0.61 g of organic polymer particles Am-8 as a white powder.

The weight of the organic polymer particles Am-8 was larger than theweight of the organic polymer particles Hy-1. Comparison of the infraredabsorption spectrum of the organic polymer particles Am-8 (afterethylenediamine treatment) with the infrared absorption spectrum of theorganic polymer particles Hy-1 (before ethylenediamine treatment)indicates that in the infrared absorption spectrum of the organicpolymer particles Am-8, a peak originating from an epoxy group, whichwas observed around 900 cm⁻¹ of the infrared absorption spectrum of theorganic polymer particles Hy-1, disappeared and, instead, peaks typicalto primary amine appeared around 3,300 cm⁻¹ and 3,500 cm⁻¹. Based on theresults, the organic polymer particles Am-8 were confirmed to have anamino group introduced into the organic polymer particles Hy-1.

6 mg of N-hydroxysuccinic acid imide was added to 3 g of a 1% aqueoussolution of CMC-1 which was obtained in Synthetic Example 14, and themixture was stirred at room temperature for 10 minutes. Next, thesolution was cooled with ice and 20 mg of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added.The mixture was stirred under ice cooling for 30 minutes. Next, 30.1 mgof the above organic polymer particles Am-8 was added to the solution.The mixture was irradiated with indirect ultrasonic radiation for 20minutes and stirred under ice cooling for 12 hours. The particles wereisolated by centrifugal separation, dispersed in purified water,isolated by centrifugal separation, and washed. This procedure wasrepeated 10 times, followed by drying to obtain 35.0 mg of carrierpolymer particle precursor pre-P-11.

In addition to the peaks originating from the organic polymer particlesAm-8 before the reaction, peaks originating from carboxymethylcellulosewere observed around 3,400 cm⁻¹ and 1,600 cm⁻¹ in the infraredabsorption spectrum of the carrier polymer particle precursor pre-P-11.Based on these results, the carrier polymer particle precursor pre-P-11was confirmed to have a saccharide (carboxymethylcellulose) bonded tothe organic polymer particles Am-8.

An operation of dispersing the carrier polymer particle precursorpre-P-11 (20.2 mg) in 2 ml of a 0.01 M sodium hydroxide aqueous solutionand isolating the particles by centrifugation was carried out threetimes, then an operation of dispersing the particles in purified waterand isolating by centrifugal separation was carried out three times,followed by drying to obtain 18.6 mg of carrier polymer particles P-11.

The peaks originating from carboxymethylcellulose were still observedaround 3,400 cm⁻¹ and 1,600 cm⁻¹ in the infrared absorption spectrum ofthe carrier polymer particles P-11, although these peaks were weaker ascompared with those in the carrier polymer particle precursor pre-P-11before the reaction. The carrier polymer particles P-11 were thusconfirmed to have a saccharide (carboxymethylcellulose) bonded to theorganic polymer particles Am-8. The carrier polymer particles P-11 werefurther treated with a 0.01 M aqueous solution of sodium hydroxide tofind that the weight loss was less than 0.1 mg, which is a negligibleamount.

4-3-3B. Evaluation Result of Nonspecific Absorption of Protein

The nonspecific protein adsorption of the carrier polymer particles P-11was measured according the above-described method to confirm that thevalue was less than the detectable limit (0.01 ng/mg).

4-3-4. Experimental Example 11 4-3-4A. Preparation of Carrier PolymerParticles P-12

The polymer particles isolated from the dispersion liquid of organicpolymer particles A-9 by centrifugation were washed by dispersing inacetone, followed by centrifugation. This washing procedure was repeatedthree times. The resulting particles were dried. 0.50 g of the particleswere put into a 100 ml flask and 25 g of ethylenediamine was added.Particles were irradiated with indirect ultrasonic radiation for 10minutes and dispersed. The dispersion liquid was stirred at 50° C. in anitrogen atmosphere for six hours, followed by isolation of theparticles by centrifugal separation. The particles were washed fourtimes with methanol and dried to obtain 0.61 g of organic polymerparticles Am-9 as a white powder. 29.9 mg of the organic polymerparticles Am-9 was added to 3 g of a 1% aqueous solution of CMC-1 whichwas obtained in Synthetic Example 14. The mixture was irradiated withindirect supersonic wave for 20 minutes to disperse the particles. Next,the solution was cooled with ice and 20 mg of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added.The mixture was stirred under ice cooling for 12 hours. The procedure ofisolation and drying was carried out in the same manner as inExperimental Example 10 to obtain 36.2 g of carrier polymer particleprecursor pre-P-12. Next, the procedure of Experimental Example 10 wasfollowed, except for using carrier polymer particle precursor pre-P-12(20.3 mg) instead of the carrier polymer particle precursor pre-P-11 ofthe Experimental Example 10, to obtain 17.9 mg of carrier polymerparticle precursor pre-P-12.

4-3-4B. Evaluation of Nonspecific Adsorption

The nonspecific protein adsorption of the carrier polymer particles P-12was measured according the above-described method to confirm that thevalue was very low (0.02 ng/mg).

4-3-5. Comparative Example 5

The nonspecific protein adsorption of the organic polymer particles A-8was measured according the above-described method to confirm that thevalue was high (1.1 ng/mg).

4-3-6. Comparative Example 6

Commercially available standard polystyrene particles (“STADEX SC200S”manufactured by JSR Corporation) was sufficiently washed with purifiedwater and their nonspecific protein adsorption was measured accordingthe above-described method to confirm that the value was very high (20ng/mg).

4-3-7. Comparative Example 7

The nonspecific protein adsorption of the carrier polymer particleprecursor pre-P-11 was measured according the above-described method toconfirm that the value was very low (0.02 ng/mg), but not lower thanthat of the carrier polymer particles P-11, of which the nonspecificprotein adsorption was less than the detectable limit.

4-3-8. Evaluation Result of Specific Trapping Property

FIG. 4 is a photograph showing the evaluation results of the specifictrapping property (an electrophoresis pattern of proteins adsorbed onthe probe-bonded particles) of carrier polymer particles P-11 and P-12obtained respectively in Experimental Examples 10 and 11, and thecarrier polymer particle precursor pre-P-11 obtained in ComparativeExample 7.

In FIG. 4, lane 1 indicates proteins trapped by the probe-bondedparticles which were prepared by using the carrier polymer particlesP-11 of Experimental Example 10, lane 2 indicates proteins trapped bythe probe-bonded particles which were prepared by using the carrierpolymer particles P-12 of Experimental Example 11, lane 3 indicatesproteins trapped by the probe-bonded particles which were prepared byusing the carrier polymer particle precursor pre-P-11 of ComparativeExample 7, lane 4 indicates the target material (AFP) 20 ng which is acontrol, lane 5 indicates the target material (AFP) 50 ng which is acontrol, and lane 6 indicates a molecular weight marker.

It can be understood from FIG. 4 that using the probe-bonded particleswhich were prepared by using the carrier polymer particles P-11 ofExperimental Example 10, only the target material (AFP) band wascollected from the serum in an amount of 16 ng per 0.2 mg of theparticles. By using the antigen-bonded particles which were prepared byusing the polymer-bonded particles P-12 of Experimental Example 11, onlythe target material (AFP) band was collected from the serum in an amountof 12 ng per 0.2 mg of the particles. On the other hand, it wasdifficult to confirm the target AFP band in the particles of ComparativeExample 7.

As a result of the above experiments, the probe-bonded particles formedusing the carrier polymer particles of Experimental Examples 10 and 11were confirmed to exhibit only small nonspecific protein adsorption andto be able to specifically trap the target material. On the other hand,the magnetic particles of Comparative Example 7 could not specificallytrap a target material, although the particles exhibited smallnonspecific protein adsorption. It can thus be understood that theparticles with a physically-adsorbed saccharide on the surface coveredwith a saccharide, such as in the particles of Comparative Example 7,cannot exhibit sufficient nonspecific trapping performance of a targetmaterial.

1. A process for producing a magnetic particle composition comprising:chemically bonding at least one magnetic particle having a diameter of0.1 to 20 micrometers to a saccharide and chemically bonding a probe forspecifically trapping a target material to the saccharide, wherein theprobe is bonded to the saccharide of the magnetic particle aftertreatment of the magnetic particle with a basic solution.
 2. The processaccording to claim 1, wherein when chemically bonding the magneticparticles and the saccharide, the at least one magnetic particle has afirst functional group and the saccharide has a second functional group,and the at least one magnetic particle and the saccharide are chemicallybonded by reacting the first functional group and the second functionalgroup.
 3. The process according to claim 2, wherein the first functionalgroup is at least one functional group selected from the groupconsisting of a carboxyl group, an epoxy group, an amino group, and atosyl group.
 4. The process according to claim 1, wherein the saccharideis a polysaccharide.
 5. The process according to claim 1, wherein thesaccharide is carboxymethylated.
 6. The process according to claim 1,wherein the magnetic particle and the saccharide are chemically bondedby a bonding group which comprises at least one selected from the groupconsisting of an amide bond and an ester bond.
 7. The process accordingto claim 1, wherein the at least one magnetic particle is obtained by aprocess comprising polymerizing a polymer layer on a magnetic materiallayer comprising a mother particle, the mother particle comprising atleast one nuclear particle and the magnetic material layer formed on thesurface of the nuclear particle, and wherein the magnetic material layercomprises at least one of Fe₂O₃ and Fe₃O₄.
 8. The process according toclaim 1, wherein the probe is at least one probe selected from the groupconsisting of a protein, a peptide, a nucleic acid, a glycosidecompound, and a synthetic chemical material.
 9. The process according toclaim 1, wherein the saccharide and the probe are bonded by a chemicalbond selected from the group consisting of an —O— bond, an —S— bond, an—SO— bond, an —SO₂— bond, a —CO— bond, a —CO₂— bond, an —NR— bond(wherein R is an alkyl group or H), an —N⁺R²R³— bond (wherein R² and R³are individually an alkyl group or H), an —NHCO— bond, and a —PO₂— bond.10. The process according to claim 1, wherein the saccharide and theprobe are bonded by a process comprising chemically reacting afunctional group in the saccharide with a functional group in the probe.11. The process according to claim 1, wherein the functional group inthe saccharide and the functional group in the probe are at least oneselected from the group consisting of a hydroxyl group, an acyl group, amercapto group, an amino group, an amino acyl group, a carbonyl group, aformyl group, a carboxyl group, an amide group, a sulfonic group, aphosphate group, an epoxy group, a tosyl group, an azido group, a vinylgroup, and an allyl group.
 12. The process according to claim 1, whereinthe target material is a biological material.
 13. The process accordingto claim 1, wherein the biological material is at least one selectedfrom the group consisting of a protein, a peptide, a nucleic acid, acarbohydrate, a lipid, or another cellular substance.
 14. The processaccording to claim 1, wherein the other cellular substance is ablood-originating substance, a floating cell, a platelet, a erythrocyte,a leukocytes, or mixtures thereof.
 15. The process according to claim 1,wherein said particle is dispersed in a dispersion medium.
 16. A processof specific trapping comprising trapping a target material in thepresence of the magnetic particle formed by the process of claim 1.